Compositions and methods for vaccinating against HSV-2

ABSTRACT

This invention relates to therapeutic polynucleotide compositions and methods for systemic immune activation which are effective for eliciting both a systemic, non-antigen specific immune response and a strong antigen-specific immune response in mammals. The polynucleotide compositions are particularly effective for protecting mammals from herpes simplex virus (HSV), such as HSV gD2, VP11/12, and VP13/14 polypeptides.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 12/604,694 filed Oct. 23, 2009, now pending, which is a divisionalapplication of U.S. application Ser. No. 11/781,153 filed Jul. 20, 2007,now issued as U.S. Pat. No. 7,628,993 issued Dec. 8, 2009; which claimsthe benefit under 35 USC §119(e) to U.S. Application Ser. No. 60/807,911filed Jul. 20, 2006. The disclosure of each of the prior applications isconsidered part of and is incorporated by reference in the disclosure ofthis application.

GRANT INFORMATION

This invention was made with government support under Grant No. 1R41AI065015-01 awarded by National Institutes of Health, NationalInstitute of Allergy and Infectious Diseases and Grant No. AI50132awarded by National Institutes of Health. The government has certainrights in the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to prophylactic and therapeuticcompositions and methods for inducing an immune response to herpessimplex virus type 2 (HSV-2). More particularly, the invention pertainsto prophylactic and therapeutic compositions and methods for inducing animmune response in a vertebrate by introducing and expressing a DNAvaccine encoding at least one of the HSV-2 proteins such as: gD,VP11/12, VP13/14 and/or VP22.

2. Background Information

Vaccination with immunogenic proteins has eliminated or reduced theincidence of many diseases; however there are major difficulties inusing proteins associated with certain pathogens and disease states asimmunogens. Many protein antigens are not intrinsically immunogenic.More often, they are not effective as vaccines because of the manner inwhich the immune system operates.

The immune system of vertebrates consists of several interactingcomponents. The best characterized and most important parts are thehumoral and cellular (cytolytic) branches. Humoral immunity involvesantibodies, proteins which are secreted into the body fluids and whichdirectly recognize an antigen. The cellular system, in contrast, relieson special cells which recognize and kill other cells which areproducing foreign antigens. This basic functional division reflects twodifferent strategies of immune defense. Humoral immunity is mainlydirected at antigens which are exogenous to the animal whereas thecellular system responds to antigens which are actively synthesizedwithin the animal.

Antibody molecules, the effectors of humoral immunity, are secreted byspecial B lymphoid cells, B cells, in response to antigen. Antibodiescan bind to and inactivate antigen directly (neutralizing antibodies) oractivate other cells of the immune system to destroy the antigen.

Cellular immune recognition is mediated by a special class of lymphoidcells, the cytotoxic T cells. These cells do not recognize wholeantigens but instead they respond to degraded peptide fragments thereofwhich appear on the surface of the target cell bound to proteins calledclass I major histocompatibility complex (MHC) molecules. Essentiallyall nucleated cells have class I molecules. It is believed that proteinsproduced within the cell are continually degraded to peptides as part ofnormal cellular metabolism. These fragments are bound to the MHCmolecules and are transported to the cell surface. Thus the cellularimmune system is constantly monitoring the spectra of proteins producedin all cells in the body and is poised to eliminate any cells producingforeign antigens.

Vaccination is the process of preparing an animal to respond to anantigen. Vaccination is more complex than immune recognition andinvolves not only B cells and cytotoxic T cells, but other types oflymphoid cells as well. During vaccination, cells which recognize theantigen (B cells or cytotoxic T cells) are clonally expanded. Inaddition, the population of ancillary cells (helper T cells) specificfor the antigen also increase. Vaccination also involves specializedantigen presenting cells which can process the antigen and display it ina form which can stimulate one of the two pathways.

Vaccination has changed little since the time of Louis Pasteur. Aforeign antigen is introduced into an animal where it activates specificB cells by binding to surface immunoglobulins. It is also taken up byantigen processing cells, wherein it is degraded, and appears infragments on the surface of these cells bound to Class II MHC molecules.Peptides bound to class II molecules are capable of stimulating thehelper class of T cells. Both helper T cells and activated B cells arerequired to produce active humoral immunization. Cellular immunity isthought to be stimulated by a similar but less understood mechanism.

Thus two different and distinct pathways of antigen processing produceexogenous antigens bound to class II MHC molecules where they canstimulate T helper cells, as well as endogenous proteins degraded andbound to class I MHC molecules and recognized by the cytotoxic class ofT cells.

There is little or no difference in the distribution of MHC molecules.Essentially all nucleated cells express class I molecules whereas classII MHC proteins are restricted to some few types of lymphoid cells.

Normal vaccination schemes will produce a humoral immune response. Theymay also provide cytotoxic immunity. The humoral system protects avaccinated individual from subsequent challenge from a pathogen and canprevent the spread of an intracellular infection if the pathogen goesthrough an extracellular phase during its life cycle; however, it can dorelatively little to eliminate intracellular pathogens. Cytotoxicimmunity complements the humoral system by eliminating the infectedcells. Thus effective vaccination should activate both types ofimmunity.

A cytotoxic T cell response is necessary to remove intracellularpathogens, such as viruses, as well as malignant cells. It has provendifficult to present an exogenously administered antigen in adequateconcentrations in conjunction with Class I molecules to assure anadequate response. This has severely hindered the development ofvaccines against tumor-specific antigens (e.g., on breast or coloncancer cells), and against weakly immunogenic viral proteins (e.g., HIV,Herpes, non-A, non-B hepatitis, CMV and EBV).

It would be desirable to provide a cellular immune response alone inimmunizing against agents, such as viruses, for which antibodies havebeen shown to enhance infectivity. It would also be useful to providesuch a response against both chronic and latent viral infections andagainst malignant cells.

The use of synthetic peptide vaccines does not necessarily solve theseproblems because either the peptides do not readily associate withhistocompatibility molecules, have a short serum half-life, are rapidlyproteolyzed, or do not specifically localize to antigen-presentingmonocytes and macrophages. At best, all exogenously administeredantigens must compete with the universe of self-proteins for binding toantigen-presenting macrophages.

Major efforts have been mounted to elicit immune responses to poorlyimmunogenic viral proteins from the herpes viruses, non-A, non-Bhepatitis, HIV, and the like. These pathogens are difficult andhazardous to propagate in vitro. Genital herpes is a highly prevalentsexually transmitted disease worldwide, and is considered to be a majorhealth burden. The causative agent is usually herpes simplex virus type2 (HSV-2). Cellular immune responses to HSV-2 are believed to beimportant for both the prevention of disease and the control ofrecurrent disease. The HSV-2 tegument proteins VP11/12, VP13/14, VP22,and gD are respectively known as, or encoded by genes, UL46, UL47, UL49,and US6. These proteins contain human CD8+ T-cell epitopes restricted byHLA A*0101, A*0201 (×2), and B*0702, respectively.

As mentioned above, synthetic peptide vaccines corresponding toviral-encoded proteins have been made, but have severe pitfalls.Attempts have also been made to use vaccinia virus vectors to expressproteins from other viruses. However, the results have beendisappointing, since (a) recombinant vaccinia viruses may be rapidlyeliminated from the circulation in already immune individuals, and (b)the administration of complex viral antigens may induce a phenomenonknown as “antigenic competition,” in which weakly immunogenic portionsof the virus fail to elicit an immune response.

Another major problem with protein or peptide vaccines is anaphylacticreaction which can occur when injections of antigen are repeated inefforts to produce a potent immune response. In this phenomenon, IgEantibodies formed in response to the antigen cause severe and sometimesfatal allergic reactions.

Accordingly, there is a need for a method for invoking a safe andeffective immune response to a protein or polypeptide associated withherpes simplex virus type 2 (HSV-2). Moreover, there is a great need fora method that will associate these antigens with Class Ihistocompatibility antigens on the cell surface to elicit a cytotoxic Tcell response, avoid anaphylaxis and proteolysis of the material in theserum, and facilitate localization of the material to monocytes andmacrophages.

SUMMARY OF THE INVENTION

The present invention provides for DNA vaccines, some of which comprisethe HSV-2 tegument genes UL46, UL47 and UL49 and another alternativecomprises the HSV-2 gD gene all of which were individually cloned intoexpression plasmids (VR1012) and used to immunize a vertebrate. Eachanimal received three 100 μg doses of formulated DNA vaccine byintramuscular (IM) injection. Formulations based on VAXFECTIN adjuvant(an adjuvant composition comprising GAP-DMORIE and DPyPE) and poloxamerwere evaluated for their ability to boost the immune responses to theHSV DNA vaccines. Plasmid DNA formulated with PBS was used as a control.Each tegument protein DNA vaccine induced strong humoral responses.Regardless of vaccine formulation, UL49 and UL47 elicited strongercellular responses than did UL46. Poloxamer significantly boosted thecellular immune responses to the UL47 DNA vaccine, relative to the othervaccine formulations. VAXFECTIN adjuvant boosted by about two-fold theantibody responses to the UL46 and UL49 DNA vaccines.

The present invention provides a method for immunizing a vertebrateagainst herpes simplex virus, comprising obtaining a formulatedpolynucleotide, that is, a positively charged liposome containing anexpressible polynucleotide coding for an immunogenic peptide, andintroducing the formulated polynucleotide into a vertebrate, whereby theliposome is incorporated into a monocyte, a macrophage, or another cell,where an immunogenic translation product of the polynucleotide isformed, and the product is processed and presented by the cell in thecontext of the major histocompatibility complex, thereby eliciting animmune response against the immunogen. Again, the polynucleotide is DNA,although mRNA may also be used.

In another embodiment, there is provided a method for delivering apharmaceutical or immunogenic polypeptide to the interior of a cell of avertebrate in vivo comprising introducing an unformulatedpolynucleotide, that is, a preparation comprising a pharmaceuticallyacceptable injectable carrier and a polynucleotide operatively codingfor the herpes simplex virus polypeptide, into the interstitial space ofa tissue comprising the cell, whereby the polynucleotide is taken upinto the interior of the cell and has an immunogenic or pharmacologicaleffect on the vertebrate. Also provided is a method for introducing apolynucleotide into muscle cells in vivo, comprising providing acomposition comprising a polynucleotide in a pharmaceutically acceptablecarrier, and contacting the composition with muscle tissue of avertebrate in vivo, whereby the polynucleotide is introduced into musclecells of the tissue. In this embodiment, the carrier preferably isisotonic, hypotonic, or weakly hypertonic, and has a relatively lowionic strength, such as provided by a sucrose solution.

One particularly attractive aspect of the invention is a method forobtaining long term administration of a herpes simplex polypeptide to avertebrate, comprising introducing an unformulated or formulated DNAsequence operatively coding for the polypeptide interstitially intotissue of the vertebrate, whereby cells of the tissue produce thepolypeptide for at least one month or at least 3 months, more preferablyat least 6 months. In this embodiment of the invention, the cellsproducing the polypeptide are nonproliferating cells, such as musclecells.

Another method according to the invention is a method for obtainingtransitory expression of a herpes simplex polypeptide in a vertebrate,comprising introducing unformulated or formulated mRNA sequenceoperatively coding for the polypeptide interstitially into tissue of thevertebrate, whereby cells of the tissue produce the polypeptide for lessthan about 20 days, usually less than about 10 days, and often less than3 or 5 days. For many of the methods of the invention, administrationinto solid tissue is preferred.

One important aspect of the present invention is a method for treatmentof genital herpes, comprising introducing a therapeutic amount of acomposition comprising at least one polynucleotide operatively codingfor gD, VP11/12, VP13/14 and/or VP22 in a pharmaceutically acceptableinjectable carrier in vivo into muscle tissue of an animal sufferingfrom genital herpes, whereby the polynucleotide is taken up into thecells and gD, VP11/12, VP13/14 and/or VP22 is produced in vivo.Preferably, the polynucleotide is a formulated polynucleotide and thecomposition is introduced interstitially into the muscle tissue;however, an unformulated polynucleotide is also contemplated.

The present invention also includes pharmaceutical products for all ofthe uses contemplated in the methods described herein. For example,there is a pharmaceutical product, comprising unformulated or formulatedpolynucleotide, operatively coding for a herpes simplex polypeptide, inphysiologically acceptable administrate form, in a container.

In another embodiment, the invention provides a pharmaceutical product,comprising unformulated or formulated polynucleotide, operatively codingfor a herpes simplex peptide, in solution in a physiologicallyacceptable injectable carrier and suitable for introductioninterstitially into a tissue to cause cells of the tissue to express thepolypeptide, and a container enclosing the solution. The peptide may beimmunogenic and administration of the solution to a human may serve tovaccinate the human, or an animal. Similarly, the peptide may betherapeutic and administration of the solution to a vertebrate in needof therapy relating to the polypeptide will have a therapeutic effect.

Also provided by the present invention is a pharmaceutical product,comprising unformulated or formulated antisense polynucleotide, insolution in a physiologically acceptable injectable carrier and suitablefor introduction interstitially into a tissue to cause cells of thetissue to take up the polynucleotide and provide a therapeutic effect,and a container enclosing the solution.

One particularly important aspect of the invention relates to apharmaceutical product for treatment of genital herpes, comprising asterile, pharmaceutically acceptable carrier, a pharmaceuticallyeffective amount of a unformulated or formulated polynucleotideoperatively coding for at least one gD, VP11/12, VP13/14 and/or VP22protein in the carrier, and a container enclosing the carrier and thepolynucleotide in sterile fashion. Preferably, the polynucleotide isDNA.

From yet another perspective, the invention includes a pharmaceuticalproduct for use in supplying a herpes simplex polypeptide to avertebrate, comprising a pharmaceutically effective amount of aunformulated or formulated polynucleotide operatively coding for eithergD, VP11/12, VP13/14, VP22 or a combination thereof, a containerenclosing the carrier and the polynucleotide in a sterile fashion, andmeans associated with the container for permitting transfer of thepolynucleotide from the container to the interstitial space of a tissue,whereby cells of the tissue can take up and express the polynucleotide.The means for permitting such transfer can include a conventional septumthat can be penetrated, e.g., by a needle. Alternatively, when thecontainer is a syringe, the means may be considered to comprise theplunger of the syringe or a needle attached to the syringe. Containersused in the present invention will usually have at least 1, preferablyat least 5 or 10, and more preferably at least 50 or 100 micrograms ofpolynucleotide, to provide one or more unit dosages. For manyapplications, the container will have at least 500 micrograms or 1milligram, and often will contain at least 50 or 100 milligrams ofpolynucleotide.

Another aspect of the invention provides a pharmaceutical product foruse in immunizing a vertebrate, comprising a pharmaceutically effectiveamount of an unformulated or formulated polynucleotide operativelycoding for either gD, VP11/12, VP13/14, VP22 or a combination thereof, asealed container enclosing the polynucleotide in a sterile fashion, andmeans associated with the container for permitting transfer of thepolynucleotide from the container to the interstitial space of a tissue,whereby cells of the tissue can take up and express the polynucleotide.

Still another aspect of the present invention is the use of unformulatedor formulated polynucleotide operatively coding for a physiologicallyactive form of either gD, VP11/12, VP13/14, VP22 or a combinationthereof, in the preparation of a pharmaceutical for introductioninterstitially into tissue to cause cells comprising the tissue toproduce the either gD, VP11/12, VP13/14, VP22 or a combination thereoffor treatment of genital herpes.

The tissue into which the polynucleotide is introduced can be apersistent, non-dividing cell. The polynucleotide may be either a DNA orRNA sequence. When the polynucleotide is DNA, it can also be a DNAsequence which is itself non-replicating, but is inserted into aplasmid, and the plasmid further comprises a replicator. The DNA may bea sequence engineered so as not to integrate into the host cell genome.The polynucleotide sequences may code for a herpes simplex viruspolypeptide which is either contained within the cells or secretedtherefrom, or may comprise a sequence which directs the secretion of thepeptide.

The DNA sequence may also include a promoter sequence. In one preferredembodiment, the DNA sequence includes a cell-specific promoter thatpermits substantial transcription of the DNA only in predeterminedcells. The DNA may also code for a polymerase for transcribing the DNA,and may comprise recognition sites for the polymerase and the injectablepreparation may include an initial quantity of the polymerase.

In many instances, it is preferred that the polynucleotide is translatedfor a limited period of time so that the polypeptide delivery istransitory. The polypeptide may advantageously be a therapeuticpolypeptide, and may comprise an enzyme, a hormone, a lymphokine, areceptor, particularly a cell surface receptor, a regulatory protein,such as a growth factor or other regulatory agent, or any other proteinor peptide that one desires to deliver to a cell in a living vertebrateand for which corresponding DNA or mRNA can be obtained.

In preferred embodiments, the polynucleotide is introduced into muscletissue; in other embodiments the polynucleotide is incorporated intotissues of skin, brain, lung, liver, spleen or blood. The preparation isinjected into the vertebrate by a variety of routes, which may beintradermally, subdermally, intrathecally, or intravenously, or it maybe placed within cavities of the body. In a preferred embodiment, thepolynucleotide is injected intramuscularly. In still other embodiments,the preparation comprising the polynucleotide is impressed into theskin. Transdermal administration is also contemplated, as is inhalation.

In one preferred embodiment, the polynucleotide is DNA coding for both apolypeptide and a polymerase for transcribing the DNA, and the DNAincludes recognition sites for the polymerase and the injectablepreparation further includes a means for providing an initial quantityof the polymerase in the cell. The initial quantity of polymerase may bephysically present together with the DNA. Alternatively, it may beprovided by including mRNA coding therefore, which mRNA is translated bythe cell. In this embodiment of the invention, the DNA is preferably aplasmid. Preferably, the polymerase is phage T7 polymerase and therecognition site is a T7 origin of replication sequence.

In accordance with another aspect of the present invention, there isprovided a method for immunizing a vertebrate, comprising the steps ofobtaining a preparation comprising an expressible polynucleotide codingfor an immunogenic translation product (that is, either gD, VP11/12,VP13/14, VP22 or a combination thereof), and introducing the preparationinto a vertebrate wherein the translation product of the polynucleotideis formed by a cell of the vertebrate, which elicits an immune responseagainst the herpes simplex virus immunogen. In one embodiment of themethod, the injectable preparation comprises a pharmaceuticallyacceptable carrier containing an expressible polynucleotide coding foran immunogenic peptide, and on the introduction of the preparation intothe vertebrate, the polynucleotide is incorporated into a cell of thevertebrate wherein an immunogenic translation product of thepolynucleotide is formed, which elicits an immune response against theimmunogen.

In an alternative embodiment, the preparation comprises one or morecells obtained from the vertebrate and transfected in vitro with thepolynucleotide (that is, either, UL46, UL47, UL49, or US6 or acombination thereof), whereby the polynucleotide is incorporated intosaid cells, where an immunogenic translation product of thepolynucleotide is formed, and whereby on the introduction of thepreparation into the vertebrate, an immune response against theimmunogen is elicited. In any of the embodiments of the invention, theimmunogenic product may be secreted by the cells, or it may be presentedby a cell of the vertebrate in the context of the majorhistocompatibility antigens, thereby eliciting an immune responseagainst the immunogen. The method may be practiced using non-dividing,differentiated cells from the vertebrates, which cells may belymphocytes, obtained from a blood sample; alternatively, it may bepracticed using partially differentiated skin fibroblasts which arecapable of dividing. In a preferred embodiment, the method is practicedby incorporating the polynucleotide coding for an immunogenictranslation product into muscle tissue.

The method may be used to selectively elicit a humoral immune response,a cellular immune response, or a mixture of these. In embodimentswherein the cell expresses major histocompatibility complex of Class I,and the immunogenic peptide is presented in the context of the Class Icomplex, the immune response is cellular and comprises the production ofcytotoxic T-cells.

In one such embodiment, the immunogenic peptide is associated with theHSV-2 virus and is presented in the context of Class I antigens, andstimulates cytotoxic T-cells which are capable of destroying cellsinfected with the virus. A cytotoxic T-cell response may also beproduced according the method where the polynucleotide codes for eithera truncated gD, VP11/12, VP13/14, VP22 or a combination thereof antigenlacking humoral epitopes.

In another embodiment, there is provided a method of immunizing avertebrate, comprising obtaining a positively charged liposomecontaining an expressible polynucleotide coding for either gD, VP11/12,VP13/14, VP22 or a combination thereof, and introducing the liposomeinto a vertebrate, whereby the liposome is incorporated into a monocyte,a macrophage, or another cell, where an immunogenic translation productof the polynucleotide is formed, and the product is processed andpresented by the cell in the context of the major histocompatibilitycomplex, thereby eliciting an immune response against the immunogen.Again, the polynucleotide is preferably DNA, although mRNA may also beused. And as before, the method may be practiced without the liposome,utilizing just the polynucleotide in an injectable carrier.

The present invention is directed to enhancing the immune response of avertebrate or mammal in need of protection against herpes simplex virusinfection by administering in vivo, into a tissue of the vertebrate, atleast one polynucleotide, wherein the polynucleotide comprises one ormore nucleic acid fragments, where the one or more nucleic acidfragments are optionally fragments of codon-optimized coding regionsoperably encoding one or more herpes simplex virus polypeptides, orfragments, variants, or derivatives thereof. The present invention isfurther directed to enhancing the immune response of a vertebrate inneed of protection against herpes simplex virus infection byadministering, in vivo, into a tissue of the vertebrate, apolynucleotide described above plus at least one isolated herpes simplexvirus polypeptide or a fragment, a variant, or derivative thereof. Theisolated herpes simplex virus polypeptide can be, for example, apurified subunit, a recombinant protein, a viral vector expressing anisolated herpes simplex virus polypeptide, or can be an inactivated orattentuated herpes simplex virus, such as those present in conventionalherpes simplex virus vaccines. According to either method, thepolynucleotide is incorporated into the cells of the vertebrate in vivo,and an immunologically effective amount of an immunogenic epitope of theencoded herpes simplex virus polypeptide, or a fragment, variant, orderivative thereof, is produced in vivo. When utilized, an isolatedherpes simplex virus polypeptide or a fragment, variant, or derivativethereof is also administered in an immunologically effective amount

According to the present invention, the polynucleotide can beadministered either prior to, at the same time (simultaneously), orsubsequent to the administration of the isolated herpes simplex viruspolypeptide. The herpes simplex virus polypeptide or fragment, variant,or derivative thereof encoded by the polynucleotide comprises at leastone immunogenic epitope capable of eliciting an immune response toherpes simplex virus in a vertebrate. In addition, an isolated herpessimplex virus polypeptide or fragment, variant, or derivative thereof,when used, comprises at least one immunogenic epitope capable ofeliciting an immune response in a vertebrate. The herpes simplex viruspolypeptide or fragment, variant, or derivative thereof encoded by thepolynucleotide can, but need not, be the same protein or fragment,variant, or derivative thereof as the isolated herpes simplex viruspolypeptide which can be administered according to the method.

The polynucleotide of the invention can comprise a nucleic acidfragment, where the nucleic acid fragment is a fragment of acodon-optimized coding region operably encoding any herpes simplex viruspolypeptide or fragment, variant, or derivative thereof, including, butnot limited to, gD, VP 11/12, VP13/14 and/or VP22 proteins or fragments,variants or derivatives thereof. A polynucleotide of the invention canalso encode a derivative fusion protein, wherein two or more nucleicacid fragments, at least one of which encodes a herpes simplex viruspolypeptide or fragment, variant, or derivative thereof, are joined inframe to encode a single polypeptide, such as, but not limited to, gD,VP 11/12, VP13/14 and/or VP22. Additionally, a polynucleotide of theinvention can further comprise a heterologous nucleic acid or nucleicacid fragment. Such heterologous nucleic acid or nucleic acid fragmentmay encode a heterologous polypeptide fused in frame with thepolynucleotide encoding the herpes simplex virus polypeptide, e.g., ahepatitis B core protein or a secretory signal peptide. Preferably, thepolynucleotide encodes a herpes simplex virus polypeptide or fragment,variant, or derivative thereof comprising at least one immunogenicepitope of herpes simplex virus, wherein the epitope elicits a B-cell(antibody) response, a T-cell (e.g., CTL) response, or both.

Similarly, the isolated herpes simplex virus polypeptide or fragment,variant, or derivative thereof to be delivered (either a recombinantprotein, a purified subunit, or viral vector expressing an isolatedherpes simplex virus polypeptide, or in the form of an inactivatedherpes simplex virus vaccine) can be any isolated herpes simplex viruspolypeptide or fragment, variant, or derivative thereof, including butnot limited to the gD, VP 11/12, VP13/14 and/or VP22 proteins orfragments, variants or derivatives thereof. In certain embodiments, aderivative protein can be a fusion protein. In other embodiments, theisolated herpes simplex virus polypeptide or fragment, variant, orderivative thereof can be fused to a heterologous protein, e.g., asecretory signal peptide or the hepatitis B virus core protein.

Nucleic acids and fragments thereof of the present invention can bealtered from their native state in one or more of the following ways.First, a nucleic acid or fragment thereof which encodes a herpes simplexvirus polypeptide or fragment, variant, or derivative thereof can bepart or all of a codon-optimized coding region, optimized according tocodon usage in the animal in which the vaccine is to be delivered. Inaddition, a nucleic acid or fragment thereof which encodes a herpessimplex virus polypeptide can be a fragment which encodes only a portionof a full-length polypeptide, and/or can be mutated so as to, forexample, remove from the encoded polypeptide non-desired protein motifspresent in the encoded polypeptide or virulence factors associated withthe encoded polypeptide. For example, the nucleic acid sequence could bemutated so as not to encode a membrane anchoring region that wouldprevent release of the polypeptide from the cell. Upon delivery, thepolynucleotide of the invention is incorporated into the cells of thevertebrate in vivo, and a prophylactically or therapeutically effectiveamount of an immunologic epitope of a herpes simplex virus is producedin vivo.

The invention further provides immunogenic compositions comprising atleast one polynucleotide, wherein the polynucleotide comprises one ormore nucleic acid fragments, where each nucleic acid fragment is afragment of a codon-optimized coding region encoding a herpes simplexvirus polypeptide or a fragment, a variant, or a derivative thereof; andimmunogenic compositions comprising a polynucleotide as described aboveand at least one isolated herpes simplex virus polypeptide or afragment, a variant, or derivative thereof. Such compositions canfurther comprise, for example, carriers, excipients, transfectionfacilitating agents, and/or adjuvants as described herein.

The immunogenic compositions comprising a polynucleotide and an isolatedherpes simplex virus polypeptide or fragment, variant, or derivativethereof as described above can be provided so that the polynucleotideand protein formulation are administered separately, for example, whenthe polynucleotide portion of the composition is administered prior (orsubsequent) to the isolated herpes simplex virus polypeptide portion ofthe composition. Alternatively, immunogenic compositions comprising thepolynucleotide and the isolated herpes simplex virus polypeptide orfragment, variant, or derivative thereof can be provided as a singleformulation, comprising both the polynucleotide and the protein, forexample, when the polynucleotide and the protein are administeredsimultaneously. In another alternative, the polynucleotide portion ofthe composition and the isolated herpes simplex virus polypeptideportion of the composition can be provided simultaneously, but inseparate formulations.

Compositions comprising at least one polynucleotide comprising one ormore nucleic acid fragments, where each nucleic acid fragment isoptionally a fragment of a codon-optimized coding region operablyencoding a herpes simplex virus polypeptide or fragment, variant, orderivative thereof together with and one or more isolated herpes simplexvirus polypeptides or fragments, variants or derivatives thereof (aseither a recombinant protein, a purified subunit, a viral vectorexpressing the protein, or in the form of an inactivated or attenuatedherpes simplex virus vaccine) will be referred to herein as“combinatorial polynucleotide (e.g., DNA) vaccine compositions” or“single formulation heterologous prime-boost vaccine compositions.”

The compositions of the invention can be univalent, bivalent, trivalentor multivalent. A univalent composition will comprise only onepolynucleotide comprising a nucleic acid fragment, where the nucleicacid fragment is optionally a fragment of a codon-optimized codingregion encoding a herpes simplex virus polypeptide or a fragment,variant, or derivative thereof, and optionally the same herpes simplexvirus polypeptide or a fragment, variant, or derivative thereof inisolated form. In a single formulation heterologous prime-boost vaccinecomposition, a univalent composition can include a polynucleotidecomprising a nucleic acid fragment, where the nucleic acid fragment isoptionally a fragment of a codon-optimized coding region encoding aherpes simplex virus polypeptide or a fragment, variant, or derivativethereof and an isolated polypeptide having the same antigenic region asthe polynucleotide. A bivalent composition will comprise, either inpolynucleotide or protein form, two different herpes simplex viruspolypeptides or fragments, variants, or derivatives thereof, eachcapable of eliciting an immune response. The polynucleotide(s) of thecomposition can encode two herpes simplex virus polypeptides oralternatively, the polynucleotide can encode only one herpes simplexvirus polypeptide and the second herpes simplex virus polypeptide wouldbe provided by an isolated herpes simplex virus polypeptide of theinvention as in, for example, a single formulation heterologousprime-boost vaccine composition. In the case where both herpes simplexvirus polypeptides of a bivalent composition are delivered inpolynucleotide form, the nucleic acid fragments operably encoding thoseherpes simplex virus polypeptides need not be on the samepolynucleotide, but can be on two different polynucleotides. A trivalentor further multivalent composition will comprise three herpes simplexvirus polypeptides or fragments, variants or derivatives thereof, eitherin isolated form or encoded by one or more polynucleotides of theinvention.

The present invention further provides plasmids and other polynucleotideconstructs for delivery of nucleic acid fragments of the invention to avertebrate, e.g., a human, which provide expression of herpes simplexvirus polypeptides, or fragments, variants, or derivatives thereof. Thepresent invention further provides carriers, excipients,transfection-facilitating agents, immunogenicity-enhancing agents, e.g.,adjuvants, or other agent or agents to enhance the transfection,expression or efficacy of the administered gene and its gene product.

In one embodiment, a multivalent composition comprises a singlepolynucleotide, e.g., plasmid, comprising one or more nucleic acidregions operably encoding herpes simplex virus polypeptides orfragments, variants, or derivatives thereof. Reducing the number ofpolynucleotides, e.g., plasmids in the compositions of the invention canhave significant impacts on the manufacture and release of product,thereby reducing the costs associated with manufacturing thecompositions. There are a number of approaches to include more than oneexpressed antigen coding sequence on a single plasmid. These include,for example, the use of Internal Ribosome Entry Site (IRES) sequences,dual promoters/expression cassettes, and fusion proteins.

The invention also provides methods for enhancing the immune response ofa vertebrate to herpes simplex virus infection by administering to thetissues of a vertebrate one or more polynucleotides each comprising oneor more nucleic acid fragments, where each nucleic acid fragment isoptionally a fragment of a codon-optimized coding region encoding aherpes simplex virus polypeptide or fragment, variant, or derivativethereof; and optionally administering to the tissues of the vertebrateone or more isolated herpes simplex virus polypeptides, or fragments,variants, or derivatives thereof. The isolated herpes simplex viruspolypeptide can be administered prior to, at the same time(simultaneously), or subsequent to administration of the polynucleotidesencoding herpes simplex virus polypeptides.

In addition, the invention provides consensus amino acid sequences forherpes simplex virus polypeptides, or fragments, variants or derivativesthereof, including, but not limited to, the gD, VP 11/12, VP13/14 and/orVP22 proteins or fragments, variants or derivatives thereof.Polynucleotides which encode the consensus polypeptides or fragments,variants or derivatives thereof, are also embodied in this invention.Such polynucleotides can be obtained by known methods, for example bybacktranslation of the amino acid sequence and PCR synthesis of thecorresponding polynucleotide as described below.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and form a part ofthe specifications, illustrate the preferred embodiments of the presentinvention, and together with the description serve to explain theprinciples of the invention.

FIG. 1 is a schematic representation of the VR1012DNA vaccine backboneor plasmid.

FIGS. 2A through 2C demonstrate sample data for CD4 and CD8 enrichmentby negative selection. Fractions were stained with labeled monoclonalantibody (mAb) and analyzed by flow cytometry.

FIGS. 3A through 3C illustrate comparison of codon-optimized andwild-type plasmids encoding full-length HSV-2 genes for activation ofcloned CD8+ T-cells specific for HSV-2 epitopes. Cos-7 cells weretransfected with either (“codon optimized”) vaccines, or wild-type(strain HG52) plasmids, and 50 ng/well relevant human class I HLA cDNA.These APC were incubated with CD8⁺ T cell clones known to respond to therelevant proteins. Supernatants were assayed for IFN-γ by ELISA.

FIG. 4 is a graph illustrating the reactivity of human serum pools withrecombinant HSV-2 tegument proteins, each plated at a 1:5 dilution foruse as capture antigen. Binding of human IgG from pooled sera of donorswith HSV-2, or without either HSV-1 or HSV-2 infection was detected byroutine ELISA.

FIGS. 5A through 5I contain graphs illustrating the IgG responsesinduced by HSV-2 tegument DNA vaccines in BALB/c mice detected by ELISA.Serum was collected before each immunization and at terminal sacrifice(X axis at days 0, 14, 28 and 42). Bars are geometric means. Antibodytiters (Y axis) were determined from OD₄₅₀ values.

FIGS. 6A through 6C graphically demonstrate the cellular responses toHSV-2 tegument DNA vaccines at day 42. Mice (10/group) were immunized onDays 0, 14, and 28, and splenocytes tested on Day 42. Each stacked barrepresents a single animal. Between 4 and 8 peptide pools (18-24peptides/pool) were tested per ORF. The heights of single bars indicateIFN-γ spot forming units (SFU)/10⁶ splenocytes. Note the differingY-axes. If the SFU were too numerous to count (TNTC), they werearbitrarily shown as 1,000.

FIG. 7 is an example of peptide truncation for minimal epitopes and CD4⁺vs. CD8⁺ responses. The epitopes begin at amino acid position 183 or 181of UL46 peptide and are 9- or 11-mers.

FIG. 8 is an example of peptide truncation for minimal epitopes and CD4⁺vs. CD8⁺ responses. The epitopes begin at amino acid position 388, 391,389 or 399 and are 11- or 13-mers.

FIG. 9 graphically demonstrates the peptide dose curve for selected UL49peptides; this region of the protein forms a CD8⁺ epitope. The peptidesare 9-, 11- or 13-mers beginning at amino acid positions 199, 200 or201.

FIG. 10 graphically demonstrates the peptide dose curve for selectedUL46 peptides; this region of the protein forms a CD4⁺ epitope. Thepeptides are 11- or 13-mers beginning at amino acid positions 388, 389,391 or 393.

FIG. 11 schematically demonstrates the identified human and H-2^(d) CD4⁺and CD8⁺ epitopes in UL46, UL47, and UL49. For H-2^(d) epitopes, barheight is proportional to EC₅₀. Footnotes 1-5 as marked are:

1 Verjans et al, J Infect Dis 2000; 182: 923-927

2 Koelle et al Proc Nat Acad Sci USA 2003; 100: 12899-12904

3 Posavad et al, J Immunol 2003; 170: 4380-4388

4 Koelle et al, J Immunol 2001; 166: 4049-4058 and

5 Koelle et al, J Virol 1998; 72: 7476-7483

FIG. 12 is the plasmid details of the present invention encoding gD,including the VR2139 plasmid construct, and the amino acid sequence (SEQID NO:1) and codon-optimized nucleic acid sequence (SEQ ID NO:2) for gD.

FIG. 13 is the plasmid details of the present invention encoding UL49,including the VR 2143 plasmid construct, and the amino acid sequence(SEQ ID NO:3) and codon-optimized nucleic acid sequence (SEQ ID NO:4)for UL 49.

FIG. 14 is the plasmid details of the present invention encoding UL47,including the VR 2144 plasmid construct, and the amino acid sequence(SEQ ID NO:5) and codon-optimized nucleic acid sequence (SEQ ID NO:6)for UL 47.

FIG. 15 is the plasmid details of the present invention encoding UL46,including the VR 2145 plasmid construct, and the amino acid sequence(SEQ ID NO:7) and codon-optimized nucleic acid sequence (SEQ ID NO:8)for UL 46.

FIG. 16 provides the codon-optimized nucleic acid sequence for truncatedHSV-2 gD (SEQ ID NO:9).

FIG. 17 provides the codon-optimized nucleic acid sequence for UL49 (SEQID NO:10).

FIG. 18 provides the codon-optimized nucleic acid sequence for UL47 (SEQID NO:11).

FIG. 19 provides the codon-optimized nucleic acid sequence for UL 46(SEQ ID NO:12).

FIGS. 20A through 20D show the immunogenicity of HSV-2 tegument DNAvaccines in BALB/c mice. Serum was collected before each immunizationand at terminal sacrifice. Top three panels show antibody titers (Yaxes) determined by ELISA against proteins made from transfected VM92cells (Kumar, S., et al., A DNA vaccine encoding the 42 kDa C-terminusof merozoite surface protein 1 of Plasmodium falciparum inducesantibody, interferon-gamma and cytotoxic T cell responses in rhesusmonkeys: immuno-stimulatory effects of granulocyte macrophage-colonystimulating factor. Immunol Lett, 2002. 81(1): p. 13-24).Peroxidase-conjugated goat anti-mouse IgG and colorimetric detection wasused to measure mouse IgG. Each symbol represents an individual animal;solid bars are the geometric means from 10 mice per group. Titers <1:100are plotted as 100; every naive mouse had titers <1:100 at all timepoints (not shown). Antibody titers are significantly different betweeneach sequential vaccination time points (*p<0.05, **p<0.005, pairedtwo-tailed t-test). FIG. 20D shows that the antibody response is againstcrude mixed native HSV-2 proteins at day 42.

FIGS. 21A through 21J show that T cells specific for tegument proteinshave high avidity. Splenocytes were pooled from 2-3 immunized mice andtested by IFN-γ ELISPOT with 13-amino acid and shorter peptides.Peptides were titrated in 10-fold dilutions from 10 μM to 10⁻⁶ μM. Ingeneral, responder cells reacting to CD8⁺ epitopes showed higher aviditythan cells reacting to CD4⁺ epitopes. In some cases, strong ELISPOTresponses were at 10⁻¹² M. The amino acid positions are designated foreach peptide.

FIGS. 22A through 22C show the detection of tegument-specific CD8+T-cells by intracellular cytokine cytometry. Splenocytes from a mousevaccinated three times with UL47 pDNA and then surviving challenge withvirulent HSV-2 were harvested 8 weeks later and stimulated with a poolof five optimal UL47 CD8 peptides at 1 μM each (FIG. 22A). FIG. 22C issame mouse, DMSO control. FIG. 22B is naive mouse splenocytes stimulatedwith UL47 CD8 epitope peptide pool.

FIG. 23 provides that splenocytes from tk⁻-HSV-2-infected BALB/c micerecognize tegument protein epitopes. Mice were challenged with 4×10⁷ pfutk⁻-HSV-2 five days after Depo-provera. Cells at day 14 were testing inIFN-γ ELISPOT with CD8 peptide epitopes. A previously described ICP27CD8⁺ epitope is the positive control (Haynes, J., Arlington J, Dong L,Braun R P, Payne L G, Potent protective cellular immune responsesgenerated by a DNA vaccine encoding HSV-2 ICP27 and the E. coli heatlabile enterotoxin. Vaccine, 2006. 24(23): p. 5016-26)

FIGS. 24A through 24C establish that tegument vaccines are beneficial inan HSV-2 intravaginal challenge model. Groups of 10 mice were challengedwith 50×LD₅₀ of HSV-2 strain 186 and observed for 14 days. FIG. 24A:mortality. FIG. 24B: mean intravaginal HSV-2 DNA copy numbers. FIG. 24C:Clinical scores in surviving animals.

FIGS. 25A and 25B show the immune responses to pDNA vaccine VR2139encoding gD₂ amino acid positions 1-340 administered IM to BALB/c micewith VAXFECTIN adjuvant. IgG titers by ELISA before each vaccine and atday 42 (FIG. 25A). Raw IFN-γ sfu/million splenocytes on day 42 (FIG.25B) using pooled overlapping gD₂ peptides as antigen. Each dot is anindividual mouse (n=9) and bars are mean.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention requires obtaining a formulated orunformulated polynucleotide operatively coding for a polypeptide forincorporation into vertebrate cells. A polynucleotide operatively codesfor a polypeptide when it has all the genetic information necessary forexpression by a target cell, such as promoters and the like. Thesepolynucleotides can be administered to the vertebrate by any method thatdelivers injectable materials to cells of the vertebrate, such as byinjection into the interstitial space of tissues such as muscles orskin, introduction into the circulation or into body cavities or byinhalation or insufflation. A formulated polynucleotide is injected orotherwise delivered to a vertebrate with a pharmaceutically acceptablelipid or liposome, for example, when the polynucleotide is to beassociated with a liposome, it requires a material for formingliposomes, preferably cationic or positively charged liposomes, andrequires that liposomal preparations be made from these materials. Withthe liposomal material in hand, the polynucleotide may advantageously beused to transfect cells in vitro for use as immunizing agents, or toadminister polynucleotides into bodily sites where liposomes may betaken up by phagocytic cells.

Alternatively an unformulated polynucleotide is injected or otherwisedelivered to the animal with a pharmaceutically acceptable liquidcarrier. For all applications, the liquid carrier is aqueous or partlyaqueous, comprising sterile, pyrogen-free water. The pH of thepreparation is suitably adjusted and buffered.

Polynucleotide Materials

The formulated or unformulated polynucleotide materials used accordingto the methods of the invention comprise DNA and RNA sequences or DNAand RNA sequences coding for either gD, VP11/12, VP13/14, VP22 or acombination thereof. (See U.S. Pat. Nos. 6,413,518; 6,855,317; and7,037,509; and U.S. Patent Publication US2006/0216304). Thesepolynucleotide sequences are unformulated in the sense that they arefree from any delivery vehicle that can act to facilitate entry into thecell, for example, the polynucleotide sequences are free of viralsequences, particularly any viral particles which may carry geneticinformation. Alternatively, these polynucleotide sequences areformulated with a material which promotes transfection, such asliposomal formulations, charged lipids such as, but not limited to,LIPOFECTIN reagent (cationic lipidN-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammonium chloride (DOTMA)and dioleoyl phophotidylethanolamine (DOPE)), or VAXFECTIN adjuvantdisclosed in U.S. Pat. No. 7,105,574.

The DNA sequences used in these methods can be those sequences which donot integrate into the genome of the host cell. These may benon-replicating DNA sequences, or specific replicating sequencesgenetically engineered to lack the genome-integration ability.

The polynucleotide sequences of the invention are DNA or RNA sequencesof either HSV-2 proteins gD, VP11/12, VP13/14, VP22 or a combinationthereof. The polynucleotides of the invention also can code fortherapeutic polypeptides. A polypeptide is understood to be anytranslation product of a polynucleotide regardless of size, and whetherglycosylated or not. Therapeutic polypeptides include as a primaryexample, those polypeptides that can compensate for defective ordeficient species in an animal, or those that act through toxic effectsto limit or remove harmful cells from the body. Polynucleotide sequencesof the invention preferably code for either gD, VP11/12, VP13/14, VP22or a combination thereof, and these sequences may be used in associationwith other polynucleotide sequences coding for regulatory proteins thatcontrol the expression of these polypeptides. The regulatory protein canact by binding to genomic DNA so as to regulate its transcription;alternatively, it can act by binding to messenger RNA to increase ordecrease its stability or translation efficiency.

Where the polynucleotide is DNA, promoters suitable for use in variousvertebrate systems are well known. For example, for use in murinesystems, suitable strong promoters include RSV LTR, MPSV LTR, SV40 IEP,and metallothionein promoter. In humans, on the other hand, promoterssuch as CMV IEP may advantageously be used. All forms of DNA, whetherreplicating or non-replicating, which do not become integrated into thegenome, and which are expressible, are within the methods contemplatedby the invention.

With the availability of automated nucleic acid synthesis equipment,both DNA and RNA can be synthesized directly when the nucleotidesequence is known or by a combination of PCR cloning and fermentation.Moreover, when the sequence of the desired polypeptide is known, asuitable coding sequence for the polynucleotide can be inferred.

When the polynucleotide is mRNA, it can be readily prepared from thecorresponding DNA in vitro. For example, conventional techniques utilizephage RNA polymerases SP6, T3, or T7 to prepare mRNA from DNA templatesin the presence of the individual ribonucleoside triphosphates. Anappropriate phage promoter, such as a T7 origin of replication site isplaced in the template DNA immediately upstream of the gene to betranscribed. Systems utilizing T7 in this manner are well known, and aredescribed in the literature, e.g., in Current Protocols in MolecularBiology, §3.8 (vol. 1 1988).

DNA and mRNA Vaccines

According to the methods of the invention, both expressible DNA and mRNAcan be delivered to cells to form therein a polypeptide translationproduct. If the nucleic acids contain the proper control sequences, theywill direct the synthesis of relatively large amounts of either gD,VP11/12, VP13/14, VP22 or a combination thereof. When the DNA and mRNAdelivered to the cells code either gD, VP11/12, VP13/14, VP22 or acombination thereof, the methods can be applied to achieve improved andmore effective immunity. Since the immune systems of all vertebratesoperate similarly, the applications described can be implemented in allvertebrate systems, comprising mammalian and avian species, as well asfish.

The methods of the invention may be applied by direct injection of thepolynucleotide into cells of the animal in vivo, or by in vitrotransfection of some of the animal cells which are then re-introducedinto the animal body.

The polynucleotides may be delivered to various cells of the animalbody, including muscle, skin, brain, lung, liver, spleen, or to thecells of the blood. Delivery of the polynucleotides directly in vivo ispreferably to the cells of muscle or skin. The polynucleotides may beinjected into muscle or skin using an injection syringe. They may alsobe delivered into muscle or skin using a vaccine gun.

It has recently been shown that cationic lipids can be used tofacilitate the transfection of cells in certain applications,particularly in vitro transfection. Cationic lipid based transfectiontechnology is preferred over other methods; it is more efficient andconvenient than calcium phosphate, DEAE dextran or electroporationmethods, and retrovirus mediated transfection, as discussed previously,can lead to integration events in the host cell genome that result inoncogene activation or other undesirable consequences. The knowledgethat cationic lipid technology works with messenger RNA is a furtheradvantage to this approach, because RNA is turned over rapidly byintracellular nucleases and is not integrated into the host genome. Atransfection system that results in high levels of reversible expressionis preferred to alternative methodology requiring selection andexpansion of stably transformed clones because many of the desiredprimary target cells do not rapidly divide in culture.

The ability to transfect cells at high efficiency with cationicliposomes provides an alternative method for immunization. The gene foran antigen is introduced into cells which have been removed from ananimal. The transfected cells, now expressing the antigen, arereinjected into the animal where the immune system can respond to the(now) endogenous antigen. The process can possibly be enhanced bycoinjection of either an adjuvant or lymphokines to further stimulatethe lymphoid cells.

Vaccination with nucleic acids containing either gD, VP11/12, VP13/14,VP22 or a combination thereof provides a way to specifically target thecellular immune response. Cells expressing at least one gD, VP11/12,VP13/14, and/or VP22 proteins which are secreted will enter the normalantigen processing pathways and produce both a humoral and cytotoxicresponse. The response to proteins which are not secreted is moreselective. Non-secreted proteins synthesized in cells expressing onlyclass I MHC molecules are expected to produce only a cytotoxicvaccination. Expression of the same antigen in cells bearing both classI and class II molecules may produce a more vigorous response bystimulating both cytotoxic and helper T cells. Enhancement of the immuneresponse may also be possible by injecting the gene for either gD,VP11/12, VP13/14, VP22 or a combination thereof along with a peptidefragment of the protein. The antigen is presented via class I MHCmolecules to the cellular immune system while the peptide is presentedvia class II MHC molecules to stimulate helper T cells. In any case,this method provides a way to stimulate and modulate the immune responsein a way which has not previously been possible.

Liposome-Forming Materials

Liposomes are unilamellar or multilamellar vesicles, having a membraneportion formed of lipophilic material and an interior aqueous portion.The aqueous portion is used in the present invention to contain thepolynucleotide material to be delivered to the target cell. It ispreferred that the liposome forming materials used herein have acationic group, such as a quaternary ammonium group, and one or morelipophilic groups, such as saturated or unsaturated alkyl groups havingfrom about 6 to about 30 carbon atoms. One group of suitable materialsis described in European Patent Publication No. 0187702 and isincorporated herein by reference. These compounds may be prepared asdetailed in the above-identified patent application; alternatively, atleast one of these compounds,N-(2,3-di-(9-(Z)-octadecenyloxy))-prop-1-yl-N,N,N-trimethylammoniumchloride (DOTMA), is commercially available from Bethesda ResearchLaboratories (BRL), Gaithersburg, Md. 20877, USA.

Moreover, many suitable liposome-forming cationic lipid compounds aredescribed in the literature. See, e.g., L. Stamatatos, et al.,Biochemistry, 27:3917-3925 (1988); H. Eibl, et al, BiophysicalChemistry, 10:261-271 (1979).

Liposome Preparation

Suitable liposomes for use in the present invention are commerciallyavailable. DOTMA liposomes, for example, are available under thetrademark Lipofectin from Bethesda Research Labs, Gaithersburg, Md.

Alternatively, liposomes can be prepared from readily-available orfreshly synthesized starting materials of the type previously described.Preparation of DOTMA liposomes is explained in the literature, see,e.g., p. Felgner, et al., Proc. Nat'l Acad. Sci. USA, 84:7413-7417.Similar methods can be used to prepare liposomes from other cationiclipid materials. Moreover, conventional liposome forming materials canbe used to prepare liposomes having negative charge or neutral charge.Such materials include phosphatidylcholine, cholesterol,phosphatidyl-ethanolamine, and the like. These materials can alsoadvantageously be mixed with the DOTAP or DOTMA starting materials inratios from 0% to about 75%.

Conventional methods can be used to prepare other, noncationicliposomes. These liposomes do not fuse with cell walls as readily ascationic liposomes. However, they are taken up by macrophages in vivo,and are thus particularly effective for delivery of polynucleotide tothese cells. For example, commercially dioleoyl-phosphatidyl choline(DOPC), dioleoylphosphatidyl glycerol (DOPG), and dioleoylphosphatidylethanolamine (DOPE) can be used in various combinations to makeconventional liposomes, with or without the addition of cholesterol.Thus, for example, DOPG/DOPC vesicles can be prepared by drying 50 mgeach of DOPG and DOPC under a stream of nitrogen gas into a sonicationvial. The sample is placed under a vacuum pump overnight and is hydratedthe following day with deionized water. The sample is then sonicated for2 hours in a capped vial, using a Heat Systems model 350 sonicatorequipped with an inverted cup (bath type) probe at the maximum settingwhile the bath is circulated at 15° C. Alternatively, negatively chargedvesicles can be prepared without sonication to produce multilamellarvesicles or by extrusion through nucleopore membranes to produceunilamellar vesicles of discrete size. Other methods are known andavailable to those of skill in the art.

Therapeutic Formulations Polynucleotide Salts

Administration of pharmaceutically acceptable salts of thepolynucleotides described herein is included within the scope of theinvention. Such salts may be prepared from pharmaceutically acceptablenon-toxic bases including organic bases and inorganic bases. Saltsderived from inorganic bases include sodium, potassium, lithium,ammonium, calcium, magnesium, and the like. Salts derived frompharmaceutically acceptable organic non-toxic bases include salts ofprimary, secondary, and tertiary amines, basic amino acids, and thelike. For a helpful discussion of pharmaceutical salts, see S. M. Bergeet al., Journal of Pharmaceutical Sciences 66:1-19 (1977).

Polynucleotides for injection, a preferred route of delivery, may beprepared in unit dosage form in ampules, or in multidose containers. Thepolynucleotides may be present in such forms as suspensions, solutions,or emulsions in oily or preferably aqueous vehicles. Alternatively, thepolynucleotide salt may be in lyophilized form for reconstitution, atthe time of delivery, with a suitable vehicle, such as sterilepyrogen-free water. Both liquid as well as lyophilized forms that are tobe reconstituted will comprise agents, preferably buffers, in amountsnecessary to suitably adjust the pH of the injected solution. For anyparenteral use, particularly if the formulation is to be administeredintravenously, the total concentration of solutes should be controlledto make the preparation isotonic, hypotonic, or weakly hypertonic.Nonionic materials, such as sugars, are preferred for adjustingtonicity, and sucrose is particularly preferred. Any of these forms mayfurther comprise suitable formulatory agents, such as starch or sugar,glycerol or saline. The compositions per unit dosage, whether liquid orsolid, may contain from 0.1% to 99% of polynucleotide material.

The units dosage ampules or multidose containers, in which thepolynucleotides are packaged prior to use, may comprise an hermeticallysealed container enclosing an amount of polynucleotide or solutioncontaining a polynucleotide suitable for a pharmaceutically effectivedose thereof, or multiples of an effective dose. The polynucleotide ispackaged as a sterile formulation, and the hermetically sealed containeris designed to preserve sterility of the formulation until use.

The container in which the polynucleotide is packaged is labeled, andthe label bears a notice in the form prescribed by a governmentalagency, for example the Food and Drug Administration, which notice isreflective of approval by the agency under Federal law, of themanufacture, use, or sale of the polynucleotide material therein forhuman administration.

Federal law requires that the use of pharmaceutical agents in thetherapy of humans be approved by an agency of the Federal government.Responsibility for enforcement is the responsibility of the Food andDrug Administration, which issues appropriate regulations for securingsuch approval, detailed in 21 U.S.C. §§301-392. Regulation for biologicmaterial, comprising products made from the tissues of animals isprovided under 42 U.S.C. §262. Similar approval is required by mostforeign countries. Regulations vary from country to country, but theindividual procedures are well known to those in the art.

Dosage and Route of Administration

The dosage to be administered depends to a large extent on the conditionand size of the subject being treated as well as the frequency oftreatment and the route of administration. Regimens for continuingtherapy, including dose and frequency may be guided by the initialresponse and clinical judgment. The parenteral route of injection intothe interstitial space of tissues is preferred, although otherparenteral routes, such as inhalation of an aerosol formulation, may berequired in specific administration, as for example to the mucousmembranes of the nose, throat, bronchial tissues or lungs.

In preferred protocols, a formulation comprising the nakedpolynucleotide in an aqueous carrier is injected into tissue in amountsof from 10 μl per site to about 1 ml per site. The concentration ofpolynucleotide in the formulation is from about 0.1 μg/ml to about 20mg/ml.

The present invention is directed to compositions and methods forenhancing the immune response of a vertebrate in need of protectionagainst herpes simplex virus infection by administering in vivo, into atissue of a vertebrate, at least one polynucleotide comprising one ormore nucleic acid fragments, where each nucleic acid fragment isoptionally a fragment of a codon-optimized coding region operablyencoding a herpes simplex virus polypeptide, or a fragment, variant, orderivative thereof in cells of the vertebrate in need of protection. Thepresent invention is also directed to administering in vivo, into atissue of the vertebrate the above described polynucleotide and at leastone isolated herpes simplex virus polypeptide, or a fragment, variant,or derivative thereof. The isolated herpes simplex virus polypeptide orfragment, variant, or derivative thereof can be, for example, arecombinant protein, a purified subunit protein, a protein expressed andcarried by a heterologous live or inactivated or attentuated viralvector expressing the protein, or can be an inactivated herpes simplexvirus, such as those present in conventional, commercially available,inactivated herpes simplex virus vaccines. According to either method,the polynucleotide is incorporated into the cells of the vertebrate invivo, and an immunologically effective amount of the herpes simplexprotein, or fragment or variant encoded by the polynucleotide isproduced in vivo. The isolated protein or fragment, variant, orderivative thereof is also administered in an immunologically effectiveamount. The polynucleotide can be administered to the vertebrate in needthereof either prior to, at the same time (simultaneously), orsubsequent to the administration of the isolated herpes simplex viruspolypeptide or fragment, variant, or derivative thereof.

Non-limiting examples of herpes simplex virus polypeptides within thescope of the invention include, but are not limited to, gD, VP 11/12,VP13/14 and/or VP22 polypeptides, and fragments, derivatives, andvariants thereof. Nucleotide and amino acid sequences of herpes simplexvirus polypeptides from a wide variety of herpes simplex virus types andsubtypes are known in the art.

The present invention also provides vaccine compositions and methods fordelivery of herpes simplex virus coding sequences to a vertebrate withoptimal expression and safety conferred through codon optimizationand/or other manipulations. These vaccine compositions are prepared andadministered in such a manner that the encoded gene products areoptimally expressed in the vertebrate of interest. As a result, thesecompositions and methods are useful in stimulating an immune responseagainst herpes simplex virus infection. Also included in the inventionare expression systems, delivery systems, and codon-optimized herpessimplex virus coding regions.

In a specific embodiment, the invention provides combinatorialpolynucleotide (e.g., DNA) vaccines which combine both a polynucleotidevaccine and polypeptide (e.g., either a recombinant protein, a purifiedsubunit protein, a viral vector expressing an isolated herpes simplexvirus polypeptide, or in the form of an inactivated or attenuated herpessimplex virus vaccine) vaccine in a single formulation. The singleformulation comprises a herpes simplex virus polypeptide-encodingpolynucleotide vaccine as described herein, and optionally, an effectiveamount of a desired isolated herpes simplex virus polypeptide orfragment, variant, or derivative thereof. The polypeptide may exist inany form, for example, a recombinant protein, a purified subunitprotein, a viral vector expressing an isolated herpes simplex viruspolypeptide, or in the form of an inactivated or attenuated herpessimplex virus vaccine. The herpes simplex virus polypeptide or fragment,variant, or derivative thereof encoded by the polynucleotide vaccine maybe identical to the isolated herpes simplex virus polypeptide orfragment, variant, or derivative thereof. Alternatively, the herpessimplex virus polypeptide or fragment, variant, or derivative thereofencoded by the polynucleotide may be different from the isolated herpessimplex virus polypeptide or fragment, variant, or derivative thereof.

It is to be noted that the term “a” or “an” entity refers to one or moreof that entity, for example, “a polynucleotide,” is understood torepresent one or more polynucleotides. As such, the terms “a” (or “an”),“one or more,” and “at least one” can be used interchangeably herein.

The term “polynucleotide” is intended to encompass a singular nucleicacid or nucleic acid fragment as well as plural nucleic acids or nucleicacid fragments, and refers to an isolated molecule or construct, e.g., avirus genome (e.g., a non-infectious viral genome), messenger RNA(mRNA), plasmid DNA (pDNA), or derivatives of pDNA (e.g., minicircles asdescribed in (Darquet, A-M et al., Gene Therapy 4:1341-1349 (1997))comprising a polynucleotide. A polynucleotide may comprise aconventional phosphodiester bond or a non-conventional bond (e.g., anamide bond, such as found in peptide nucleic acids (PNA)).

The terms “nucleic acid” or “nucleic acid fragment” refer to any one ormore nucleic acid segments, e.g., DNA or RNA fragments, present in apolynucleotide or construct. A nucleic acid or fragment thereof may beprovided in linear (e.g., mRNA) or circular (e.g., plasmid) form as wellas double-stranded or single-stranded forms. By “isolated” nucleic acidor polynucleotide is intended a nucleic acid molecule, DNA or RNA, whichhas been removed from its native environment. For example, a recombinantpolynucleotide contained in a vector is considered isolated for thepurposes of the present invention. Further examples of an isolatedpolynucleotide include recombinant polynucleotides maintained inheterologous host cells or purified (partially or substantially)polynucleotides in solution. Isolated RNA molecules include in vivo orin vitro RNA transcripts of the polynucleotides of the presentinvention. Isolated polynucleotides or nucleic acids according to thepresent invention further include such molecules produced synthetically.

As used herein, a “coding region” is a portion of nucleic acid whichconsists of codons translated into amino acids. Although a “stop codon”(TAG, TGA, or TAA) is not translated into an amino acid, it may beconsidered to be part of a coding region, but any flanking sequences,for example promoters, ribosome binding sites, transcriptionalterminators, and the like, are not part of a coding region. Two or morenucleic acids or nucleic acid fragments of the present invention can bepresent in a single polynucleotide construct, e.g., on a single plasmid,or in separate polynucleotide constructs, e.g., on separate (different)plasmids. Furthermore, any nucleic acid or nucleic acid fragment mayencode a single herpes simplex virus polypeptide or fragment,derivative, or variant thereof, e.g., or may encode more than onepolypeptide, e.g., a nucleic acid may encode two or more polypeptides.In addition, a nucleic acid may include a regulatory element such as apromoter, ribosome binding site, or a transcription terminator, or mayencode heterologous coding regions fused to the herpes simplex viruscoding region, e.g., specialized elements or motifs, such as a secretorysignal peptide or a heterologous functional domain.

The terms “fragment,” “variant,” “derivative” and “analog” whenreferring to herpes simplex virus polypeptides of the present inventioninclude any polypeptides which retain at least some of theimmunogenicity or antigenicity of the corresponding native polypeptide.Fragments of herpes simplex virus polypeptides of the present inventioninclude proteolytic fragments, deletion fragments and in particular,fragments of herpes simplex virus polypeptides which exhibit increasedsecretion from the cell or higher immunogenicity or reducedpathogenicity when delivered to an animal. Polypeptide fragments furtherinclude any portion of the polypeptide which comprises an antigenic orimmunogenic epitope of the native polypeptide, including linear as wellas three-dimensional epitopes. Variants of herpes simplex viruspolypeptides of the present invention include fragments as describedabove, and also polypeptides with altered amino acid sequences due toamino acid substitutions, deletions, or insertions. Variants may occurnaturally, such as an allelic variant. By an “allelic variant” isintended alternate forms of a gene occupying a given locus on achromosome or genome of an organism or virus. Genes II, Lewin, B., ed.,John Wiley & Sons, New York (1985). For example, as used herein,variations in a given gene product. When referring to herpes simplexvirus gD, VP 11/12, VP13/14 and/or VP22 proteins, each such protein is a“variant,” in that native herpes simplex virus strains are distinguishedby the type of proteins encoded by the virus. However, within a singlegD, VP 11/12, VP13/14 and/or VP22 variant type, further naturally ornon-naturally occurring variations such as amino acid deletions,insertions or substitutions may occur. Non-naturally occurring variantsmay be produced using art-known mutagenesis techniques. Variantpolypeptides may comprise conservative or non-conservative amino acidsubstitutions, deletions or additions. Derivatives of herpes simplexvirus polypeptides of the present invention, are polypeptides which havebeen altered so as to exhibit additional features not found on thenative polypeptide. Examples include fusion proteins. An analog isanother form of a herpes simplex virus polypeptide of the presentinvention. An example is a proprotein which can be activated by cleavageof the proprotein to produce an active mature polypeptide.

The terms “infectious polynucleotide” or “infectious nucleic acid” areintended to encompass isolated viral polynucleotides and/or nucleicacids which are solely sufficient to mediate the synthesis of completeinfectious virus particles upon uptake by permissive cells. Thus,“infectious nucleic acids” do not require pre-synthesized copies of anyof the polypeptides it encodes, e.g., viral replicases, in order toinitiate its replication cycle in a permissive host cell.

The terms “non-infectious polynucleotide” or “non-infectious nucleicacid” as defined herein are polynucleotides or nucleic acids whichcannot, without additional added materials, e.g., polypeptides, mediatethe synthesis of complete infectious virus particles upon uptake bypermissive cells. An infectious polynucleotide or nucleic acid is notmade “non-infectious” simply because it is taken up by a non-permissivecell. For example, an infectious viral polynucleotide from a virus withlimited host range is infectious if it is capable of mediating thesynthesis of complete infectious virus particles when taken up by cellsderived from a permissive host (i.e., a host permissive for the virusitself). The fact that uptake by cells derived from a non-permissivehost does not result in the synthesis of complete infectious virusparticles does not make the nucleic acid “non-infectious.” In otherwords, the term is not qualified by the nature of the host cell, thetissue type, or the species taking up the polynucleotide or nucleic acidfragment.

In some cases, an isolated infectious polynucleotide or nucleic acid mayproduce fully-infectious virus particles in a host cell population whichlacks receptors for the virus particles, i.e., is non-permissive forvirus entry. Thus viruses produced will not infect surrounding cells.However, if the supernatant containing the virus particles istransferred to cells which are permissive for the virus, infection willtake place.

The terms “replicating polynucleotide” or “replicating nucleic acid” aremeant to encompass those polynucleotides and/or nucleic acids which,upon being taken up by a permissive host cell, are capable of producingmultiple, e.g., one or more copies of the same polynucleotide or nucleicacid. Infectious polynucleotides and nucleic acids are a subset ofreplicating polynucleotides and nucleic acids; the terms are notsynonymous. For example, a defective virus genome lacking the genes forvirus coat proteins may replicate, e.g., produce multiple copies ofitself, but is NOT infectious because it is incapable of mediating thesynthesis of complete infectious virus particles unless the coatproteins, or another nucleic acid encoding the coat proteins, areexogenously provided.

In certain embodiments, the polynucleotide, nucleic acid, or nucleicacid fragment is DNA. In the case of DNA, a polynucleotide comprising anucleic acid which encodes a polypeptide normally also comprises apromoter and/or other transcription or translation control elementsoperably associated with the polypeptide-encoding nucleic acid fragment.An operable association is when a nucleic acid fragment encoding a geneproduct, e.g., a polypeptide, is associated with one or more regulatorysequences in such a way as to place expression of the gene product underthe influence or control of the regulatory sequence(s). Two DNAfragments (such as a polypeptide-encoding nucleic acid fragment and apromoter associated with the 5′ end of the nucleic acid fragment) are“operably associated” if induction of promoter function results in thetranscription of mRNA encoding the desired gene product and if thenature of the linkage between the two DNA fragments does not (1) resultin the introduction of a frame-shift mutation, (2) interfere with theability of the expression regulatory sequences to direct the expressionof the gene product, or (3) interfere with the ability of the DNAtemplate to be transcribed. Thus, a promoter region would be operablyassociated with a nucleic acid fragment encoding a polypeptide if thepromoter was capable of effecting transcription of that nucleic acidfragment. The promoter may be a cell-specific promoter that directssubstantial transcription of the DNA only in predetermined cells. Othertranscription control elements, besides a promoter, for exampleenhancers, operators, repressors, and transcription termination signals,can be operably associated with the polynucleotide to directcell-specific transcription. Suitable promoters and other transcriptioncontrol regions are disclosed herein.

A variety of transcription control regions are known to those skilled inthe art. These include, without limitation, transcription controlregions which function in vertebrate cells, such as, but not limited to,promoter and enhancer segments from cytomegaloviruses (the immediateearly promoter, in conjunction with intron-A), simian virus 40 (theearly promoter), and retroviruses (such as Rous sarcoma virus). Othertranscription control regions include those derived from vertebrategenes such as actin, heat shock protein, bovine growth hormone andrabbit β-globin, as well as other sequences capable of controlling geneexpression in eukaryotic cells. Additional suitable transcriptioncontrol regions include tissue-specific promoters and enhancers as wellas lymphokine-inducible promoters (e.g., promoters inducible byinterferons or interleukins).

Similarly, a variety of translation control elements are known to thoseof ordinary skill in the art. These include, but are not limited toribosome binding sites, translation initiation and termination codons,elements from picornaviruses (particularly an internal ribosome entrysite, or IRES, also referred to as a CITE sequence).

A DNA polynucleotide of the present invention may be a circular orlinearized plasmid or vector, or other linear DNA which may also benon-infectious and nonintegrating (i.e., does not integrate into thegenome of vertebrate cells). A linearized plasmid is a plasmid that waspreviously circular but has been linearized, for example, by digestionwith a restriction endonuclease. Linear DNA may be advantageous incertain situations as discussed, e.g., in Chemg, J. Y., et al., J.Control. Release 60:343-53 (1999), and Chen, Z. Y., et al. Mol. Ther.3:403-10 (2001). As used herein, the terms plasmid and vector can beused interchangeably.

Alternatively, DNA virus genomes may be used to administer DNApolynucleotides into vertebrate cells. In certain embodiments, a DNAvirus genome of the present invention is nonreplicative, noninfectious,and/or nonintegrating. Suitable DNA virus genomes include withoutlimitation, herpes simplex virus genomes, adenovirus genomes,adeno-associated virus genomes, and poxvirus genomes. References citingmethods for the in vivo introduction of non-infectious virus genomes tovertebrate tissues are well known to those of ordinary skill in the art.

In other embodiments, a polynucleotide of the present invention is RNA,for example, in the form of messenger RNA (mRNA). Methods forintroducing RNA sequences into vertebrate cells are described in U.S.Pat. No. 5,580,859.

Polynucleotides, nucleic acids, and nucleic acid fragments of thepresent invention may be associated with additional nucleic acids whichencode secretory or signal peptides, which direct the secretion of apolypeptide encoded by a nucleic acid fragment or polynucleotide of thepresent invention. According to the signal hypothesis, proteins secretedby mammalian cells have a signal peptide or secretory leader sequencewhich is cleaved from the mature protein once export of the growingprotein chain across the rough endoplasmic reticulum has been initiated.Those of ordinary skill in the art are aware that polypeptides secretedby vertebrate cells generally have a signal peptide fused to theN-terminus of the polypeptide, which is cleaved from the complete or“full length” polypeptide to produce a secreted or “mature” form of thepolypeptide. In certain embodiments, the native leader sequence is used,or a functional derivative of that sequence that retains the ability todirect the secretion of the polypeptide that is operably associated withit. Alternatively, a heterologous mammalian leader sequence, or afunctional derivative thereof, may be used. For example, the wild-typeleader sequence may be substituted with the leader sequence of humantissue plasminogen activator (TPA) or mouse β-glucuronidase.

In accordance with one aspect of the present invention, there isprovided a polynucleotide construct, for example, a plasmid, comprisinga nucleic acid fragment, where the nucleic acid fragment is a fragmentof a codon-optimized coding region operably encoding a herpes simplexvirus-derived polypeptide, where the coding region is optimized forexpression in vertebrate cells, of a desired vertebrate species, e.g.,humans, to be delivered to a vertebrate to be treated or immunized.Suitable herpes simplex virus polypeptides, or fragments, variants, orderivatives thereof may be derived from, but are not limited to, theherpes simplex virus gD, VP 11/12, VP13/14 and/or VP22 proteins.Additional herpes simplex virus-derived coding sequences, may also beincluded on the plasmid, or on a separate plasmid, and expressed, eitherusing native herpes simplex virus codons or codons optimized forexpression in the vertebrate to be treated or immunized. When such aplasmid encoding one or more optimized herpes simplex sequences isdelivered, in vivo to a tissue of the vertebrate to be treated orimmunized, one or more of the encoded gene products will be expressed,i.e., transcribed and translated. The level of expression of the geneproduct(s) will depend to a significant extent on the strength of theassociated promoter and the presence and activation of an associatedenhancer element, as well as the degree of optimization of the codingregion.

As used herein, the term “plasmid” refers to a construct made up ofgenetic material (i.e., nucleic acids). Typically a plasmid contains anorigin of replication which is functional in bacterial host cells, e.g.,Escherichia coli, and selectable markers for detecting bacterial hostcells comprising the plasmid. Plasmids of the present invention mayinclude genetic elements as described herein arranged such that aninserted coding sequence can be transcribed and translated in eukaryoticcells. Also, the plasmid may include a sequence from a viral nucleicacid. However, such viral sequences normally are not sufficient todirect or allow the incorporation of the plasmid into a viral particle,and the plasmid is therefore a non-viral vector. In certain embodimentsdescribed herein, a plasmid is a closed circular DNA molecule.

The term “expression” refers to the biological production of a productencoded by a coding sequence. In most cases a DNA sequence, includingthe coding sequence, is transcribed to form a messenger-RNA (mRNA). Themessenger-RNA is then translated to form a polypeptide product which hasa relevant biological activity. Also, the process of expression mayinvolve further processing steps to the RNA product of transcription,such as splicing to remove introns, and/or post-translational processingof a polypeptide product.

As used herein, the term “polypeptide” is intended to encompass asingular “polypeptide” as well as plural “polypeptides,” and comprisesany chain or chains of two or more amino acids. Thus, as used herein,terms including, but not limited to “peptide,” “dipeptide,”“tripeptide,” “protein,” “amino acid chain,” or any other term used torefer to a chain or chains of two or more amino acids, are included inthe definition of a “polypeptide,” and the term “polypeptide” can beused instead of, or interchangeably with any of these terms. The termfurther includes polypeptides which have undergone post-translationalmodifications, for example, glycosylation, acetylation, phosphorylation,amidation, derivatization by known protecting/blocking groups,proteolytic cleavage, or modification by non-naturally occurring aminoacids.

Also included as polypeptides of the present invention are fragments,derivatives, analogs, or variants of the foregoing polypeptides, and anycombination thereof. Polypeptides, and fragments, derivatives, analogs,or variants thereof of the present invention can be antigenic andimmunogenic polypeptides related to herpes simplex virus polypeptides,which are used to prevent or treat, i.e., cure, ameliorate, lessen theseverity of, or prevent or reduce contagion of infectious disease causedby the herpes simplex virus.

As used herein, an “antigenic polypeptide” or an “immunogenicpolypeptide” is a polypeptide which, when introduced into a vertebrate,reacts with the vertebrate's immune system molecules, i.e., isantigenic, and/or induces an immune response in the vertebrate, i.e., isimmunogenic. It is quite likely that an immunogenic polypeptide willalso be antigenic, but an antigenic polypeptide, because of its size orconformation, may not necessarily be immunogenic. Examples of antigenicand immunogenic polypeptides of the present invention include, but arenot limited to, e.g., gD, VP 11/12, VP13/14 and/or VP22 or fragments orvariants thereof, or any of the foregoing polypeptides or fragmentsfused to a heterologous polypeptide, for example, a hepatitis B coreantigen. Isolated antigenic and immunogenic polypeptides of the presentinvention in addition to those encoded by polynucleotides of theinvention, may be provided as a recombinant protein, a purified subunit,a viral vector expressing the protein, or may be provided in the form ofan inactivated herpes simplex virus vaccine, e.g., a live-attenuatedvirus vaccine, a heat-killed virus vaccine, etc.

Immunospecific binding excludes non-specific binding but does notexclude cross-reactivity with other antigens. Where all immunogenicepitopes are antigenic, antigenic epitopes need not be immunogenic.

By an “isolated” herpes simplex virus polypeptide or a fragment,variant, or derivative thereof is intended a herpes simplex viruspolypeptide or protein that is not in its natural form. No particularlevel of purification is required. For example, an isolated herpessimplex virus polypeptide can be removed from its native or naturalenvironment. Recombinantly produced herpes simplex virus polypeptidesand proteins expressed in host cells are considered isolated forpurposed of the invention, as are native or recombinant herpes simplexvirus polypeptides which have been separated, fractionated, or partiallyor substantially purified by any suitable technique, including theseparation of herpes simplex virus virions from eggs or culture cells inwhich they have been propagated. In addition, an isolated herpes simplexvirus polypeptide or protein can be provided as a live or inactivatedviral vector expressing an isolated herpes simplex virus polypeptide andcan include those found in inactivated herpes simplex virus vaccinecompositions. Thus, isolated herpes simplex virus polypeptides andproteins can be provided as, for example, recombinant herpes simplexvirus polypeptides, a purified subunit of herpes simplex virus, a viralvector expressing an isolated herpes simplex virus polypeptide, or inthe form of an inactivated or attenuated herpes simplex virus vaccine.

The term “epitopes,” as used herein, refers to portions of a polypeptidehaving antigenic or immunogenic activity in a vertebrate, for example ahuman. An “immunogenic epitope,” as used herein, is defined as a portionof a protein that elicits an immune response in an animal, as determinedby any method known in the art. The term “antigenic epitope,” as usedherein, is defined as a portion of a protein to which an antibody orT-cell receptor can immunospecifically bind as determined by any methodwell known in the art.

The term “immunogenic carrier” as used herein refers to a firstpolypeptide or fragment, variant, or derivative thereof which enhancesthe immunogenicity of a second polypeptide or fragment, variant, orderivative thereof. Typically, an “immunogenic carrier” is fused to orconjugated to the desired polypeptide or fragment thereof. An example ofan “immunogenic carrier” is a recombinant hepatitis B core antigenexpressing, as a surface epitope, an immunogenic epitope of interest.See, e.g., European Patent No. EP 0385610 B1.

In the present invention, antigenic epitopes preferably contain asequence of at least 4, at least 5, at least 6, at least 7, at least 8,at least 9, at least 10, at least 15, at least 20, at least 25, orbetween about 8 to about 30 amino acids contained within the amino acidsequence of a herpes simplex virus polypeptide of the invention, e.g.,an gD, VP 11/12, VP13/14 and/or VP22 polypeptide. Certain polypeptidescomprising immunogenic or antigenic epitopes are at least 5, 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 aminoacid residues in length. Antigenic as well as immunogenic epitopes maybe linear, i.e., be comprised of contiguous amino acids in apolypeptide, or may be three dimensional, i.e., where an epitope iscomprised of non-contiguous amino acids which come together due to thesecondary or tertiary structure of the polypeptide, thereby forming anepitope.

As to the selection of peptides or polypeptides bearing an antigenicepitope (e.g., that contain a region of a protein molecule to which anantibody or T cell receptor can bind), it is well known in that art thatrelatively short synthetic peptides that mimic part of a proteinsequence are routinely capable of eliciting an antiserum that reactswith the partially mimicked protein. See, e.g., Sutcliffe, J. G., etal., Science 219:660-666 (1983).

Peptides capable of eliciting an immunogenic response are frequentlyrepresented in the primary sequence of a protein, can be characterizedby a set of simple chemical rules, and are confined neither toimmunodominant regions of intact proteins nor to the amino or carboxylterminals. Peptides that are extremely hydrophobic and those of six orfewer residues generally are ineffective at inducing antibodies thatbind to the mimicked protein; longer peptides, especially thosecontaining proline residues, usually are effective. Sutcliffe et al.,supra, at 661.

Codon Optimization

“Codon optimization” is defined as modifying a nucleic acid sequence forenhanced expression in the cells of the vertebrate of interest, e.g.human, by replacing at least one, more than one, or a significantnumber, of codons of the native sequence with codons that are morefrequently or most frequently used in the genes of that vertebrate.Various species exhibit particular bias for certain codons of aparticular amino acid.

In one aspect, the present invention relates to polynucleotidescomprising nucleic acid fragments of codon-optimized coding regionswhich encode herpes simplex virus polypeptides, or fragments, variants,or derivatives thereof, with the codon usage adapted for optimizedexpression in the cells of a given vertebrate, e.g., humans. Thesepolynucleotides are prepared by incorporating codons preferred for usein the genes of the vertebrate of interest into the DNA sequence. Alsoprovided are polynucleotide expression constructs, vectors, and hostcells comprising nucleic acid fragments of codon-optimized codingregions which encode herpes simplex virus polypeptides, and fragments,variants, or derivatives thereof, and various methods of using thepolynucleotide expression constructs, vectors, host cells to treat orprevent herpes simplex disease in a vertebrate.

As used herein the term “codon-optimized coding region” means a nucleicacid coding region that has been adapted for expression in the cells ofa given vertebrate by replacing at least one, or more than one, or asignificant number, of codons with one or more codons that are morefrequently used in the genes of that vertebrate.

Deviations in the nucleotide sequence that comprise the codons encodingthe amino acids of any polypeptide chain allow for variations in thesequence coding for the gene. Since each codon consists of threenucleotides, and the nucleotides comprising DNA are restricted to fourspecific bases, there are 64 possible combinations of nucleotides, 61 ofwhich encode amino acids (the remaining three codons encode signalsending translation (stop or termination)). The “genetic code” whichshows which codons encode which amino acids is reproduced herein asTable 1. As a result, many amino acids are designated by more than onecodon. For example, the amino acids alanine and proline are coded for byfour triplets, serine and arginine by six, whereas tryptophan andmethionine are coded by just one triplet. This degeneracy allows for DNAbase composition to vary over a wide range without altering the aminoacid sequence of the proteins encoded by the DNA.

TABLE 1 The Standard Genetic Code T(U) C A G T TTT Phe  TCT Ser TAT Tyr (Y) TGT Cys  (U) (F) (S) TAC Tyr (C) TTC Phe TCC Ser TAA TerTGC Cys TTA Leu  TCA Ser TAG Ter TGA Ter (L) TCG Ser TGG Trp TTG Leu (W) C CTT Leu  CCT Pro  CAT His (H) CGT Arg  (L) (P) CAC His (R)CTC Leu CCC Pro CAA Gln (Q) CGC Arg CTA Leu CCA Pro CAG Gln  CGA ArgCTG Leu CCG Pro CGG Arg A ATT Ile  ACT Thr  AAT Asn (N) AGT Ser  (I) (T)AAC Asn (S) ATC Ile ACC Thr AAA Lys (K) AGC Ser ATA Ile ACA Thr AAG LysAGA Arg  ATG Met ACG Thr (R) (M) AGG Arg G GTT Val  GCT Ala  GAT Asp (D)GGT Gly  (V) (A) GAC Asp (G) GTC Val GCC Ala GAA Glu (E) GGC Gly GTA ValGCA Ala GAG Glu GGA Gly GTG Val GCG Ala GGG Gly

Many organisms display a bias for use of particular codons to code forinsertion of a particular amino acid in a growing peptide chain. Codonpreference or codon bias, differences in codon usage between organisms,is afforded by degeneracy of the genetic code, and is well documentedamong many organisms. Codon bias often correlates with the efficiency oftranslation of messenger RNA (mRNA), which is in turn believed to bedependent on, inter alia, the properties of the codons being translatedand the availability of particular transfer RNA (tRNA) molecules. Thepredominance of selected tRNAs in a cell is generally a reflection ofthe codons used most frequently in peptide synthesis. Accordingly, genescan be tailored for optimal gene expression in a given organism based oncodon optimization.

Given the large number of gene sequences available for a wide variety ofanimal, plant and microbial species, it is possible to calculate therelative frequencies of codon usage. Codon usage tables are readilyavailable, for example, at the “Codon Usage Database” available on theworld wide web at kazusa.or.jp/codon/ (Jul. 9, 2002), and these tablescan be adapted in a number of ways. See Nakamura, Y., et al. “Codonusage tabulated from the international DNA sequence databases: statusfor the year 2000” Nucl. Acids Res. 28:292 (2000). As examples, thecodon usage tables for human, mouse, domestic cat, and cow, calculatedfrom GenBank Release 128.0 (15 Feb. 2002), are reproduced below asTables 2-5. These Tables use mRNA nomenclature, and so instead ofthymine (T) which is found in DNA, the Tables use uracil (U) which isfound in RNA. The Tables have been adapted so that frequencies arecalculated for each amino acid, rather than for all 64 codons.

TABLE 2 Codon Usage Table for Human Genes (Homo sapiens) Amino AcidCodon Number Frequency Phe UUU 326146 0.4525 Phe UUC 394680 0.5475 Total720826 Leu UUA 139249 0.0728 Leu UUG 242151 0.1266 Leu CUU 246206 0.1287Leu CUC 374262 0.1956 Leu CUA 133980 0.0700 Leu CUG 777077 0.4062 Total1912925 Ile AUU 303721 0.3554 Ile AUC 414483 0.4850 Ile AUA 1363990.1596 Total 854603 Met AUG 1430946 1.0000 Total 1430946 Val GUU 2104230.1773 Val GUC 282445 0.2380 Val GUA 134991 0.1137 Val GUG 559044 0.4710Total 1186903 Ser UCU 282407 0.1840 Ser UCC 336349 0.2191 Ser UCA 2259630.1472 Ser UCG 86761 0.0565 Ser AGU 230047 0.1499 Ser AGC 373362 0.2433Total 1534889 Pro CCU 333705 0.2834 Pro CCC 386462 0.3281 Pro CCA 3222200.2736 Pro CCG 135317 0.1149 Total 1177704 Thr ACU 247913 0.2419 Thr ACC371420 0.3624 Thr ACA 285655 0.2787 Thr ACG 120022 0.1171 Total 1025010Ala GCU 360146 0.2637 Ala GCC 551452 0.4037 Ala GCA 308034 0.2255 AlaGCG 146233 0.1071 Total 1365865 Tyr UAU 232240 0.4347 Tyr UAC 3019780.5653 Total 534218 His CAU 201389 0.4113 His CAC 288200 0.5887 Total489589 Gln CAA 227742 0.2541 Gln CAG 668391 0.7459 Total 896133 Asn AAU322271 0.4614 Asn AAC 376210 0.5386 Total 698481 Lys AAA 462660 0.4212Lys AAG 635755 0.5788 Total 1098415 Asp GAU 430744 0.4613 Asp GAC 5029400.5387 Total 933684 Glu GAA 561277 0.4161 Glu GAG 787712 0.5839 Total1348989 Cys UGU 190962 0.4468 Cys UGC 236400 0.5532 Total 427362 Trp UGG248083 1.0000 Total 248083 Arg CGU 90899 0.0830 Arg CGC 210931 0.1927Arg CGA 122555 0.1120 Arg CGG 228970 0.2092 Arg AGA 221221 0.2021 ArgAGG 220119 0.2011 Total 1094695 Gly GGU 209450 0.1632 Gly GGC 4413200.3438 Gly GGA 315726 0.2459 Gly GGG 317263 0.2471 Total 1283759 StopUAA 13963 Stop UAG 10631 Stop UGA 24607

TABLE 3 Codon Usage Table for Mouse Genes (Mus musculus) Amino AcidCodon Number Frequency Phe UUU 150467 0.4321 Phe UUC 197795 0.5679 Total348262 Leu UUA 55635 0.0625 Leu UUG 116210 0.1306 Leu CUU 114699 0.1289Leu CUC 179248 0.2015 Leu CUA 69237 0.0778 Leu CUG 354743 0.3987 Total889772 Ile AUU 137513 0.3367 Ile AUC 208533 0.5106 Ile AUA 62349 0.1527Total 408395 Met AUG 1204546 1.0000 Total 1204546 Val GUU 93754 0.1673Val GUC 140762 0.2513 Val GUA 64417 0.1150 Val GUG 261308 0.4664 Total560241 Ser UCU 139576 0.1936 Ser UCC 160313 0.2224 Ser UCA 100524 0.1394Ser UCG 38632 0.0536 Ser AGU 108413 0.1504 Ser AGC 173518 0.2407 Total720976 Pro CCU 162613 0.3036 Pro CCC 164796 0.3077 Pro CCA 151091 0.2821Pro CCG 57032 0.1065 Total 535532 Thr ACU 119832 0.2472 Thr ACC 1724150.3556 Thr ACA 140420 0.2896 Thr ACG 52142 0.1076 Total 484809 Ala GCU178593 0.2905 Ala GCC 236018 0.3839 Ala GCA 139697 0.2272 Ala GCG 604440.0983 Total 614752 Tyr UAU 108556 0.4219 Tyr UAC 148772 0.5781 Total257328 His CAU 88786 0.3973 His CAC 134705 0.6027 Total 223491 Gln CAA101783 0.2520 Gln CAG 302064 0.7480 Total 403847 Asn AAU 138868 0.4254Asn AAC 187541 0.5746 Total 326409 Lys AAA 188707 0.3839 Lys AAG 3027990.6161 Total 491506 Asp GAU 189372 0.4414 Asp GAC 239670 0.5586 Total429042 Glu GAA 235842 0.4015 Glu GAG 351582 0.5985 Total 587424 Cys UGU97385 0.4716 Cys UGC 109130 0.5284 Total 206515 Trp UGG 112588 1.0000Total 112588 Arg CGU 41703 0.0863 Arg CGC 86351 0.1787 Arg CGA 589280.1220 Arg CGG 92277 0.1910 Arg AGA 101029 0.2091 Arg AGG 102859 0.2129Total 483147 Gly GGU 103673 0.1750 Gly GGC 198604 0.3352 Gly GGA 1514970.2557 Gly GGG 138700 0.2341 Total 592474 Stop UAA 5499 Stop UAG 4661Stop UGA 10356

TABLE 4 Codon Usage Table for Domestic Cat Genes (Felis cattus) AminoAcid Codon Number Frequency of usage Phe UUU 1204.00 0.4039 Phe UUC1777.00 0.5961 Total 2981 Leu UUA 404.00 0.0570 Leu UUG 857.00 0.1209Leu CUU 791.00 0.1116 Leu CUC 1513.00 0.2135 Leu CUA 488.00 0.0688 LeuCUG 3035.00 0.4282 Total 7088 Ile AUU 1018.00 0.2984 Ile AUC 1835.000.5380 Ile AUA 558.00 0.1636 Total 3411 Met AUG 1553.00 0.0036 Total1553 Val GUU 696.00 0.1512 Val GUC 1279.00 0.2779 Val GUA 463.00 0.1006Val GUG 2164.00 0.4702 Total 4602 Ser UCU 940.00 0.1875 Ser UCC 1260.000.2513 Ser UCA 608.00 0.1213 Ser UCG 332.00 0.0662 Ser AGU 672.00 0.1340Ser AGC 1202.00 0.2397 Total 5014 Pro CCU 958.00 0.2626 Pro CCC 1375.000.3769 Pro CCA 850.00 0.2330 Pro CCG 465.00 0.1275 Total 3648 Thr ACU822.00 0.2127 Thr ACC 1574.00 0.4072 Thr ACA 903.00 0.2336 Thr ACG566.00 0.1464 Total 3865 Ala GCU 1129.00 0.2496 Ala GCC 1951.00 0.4313Ala GCA 883.00 0.1952 Ala GCG 561.00 0.1240 Total 4524 Tyr UAU 837.000.3779 Tyr UAC 1378.00 0.6221 Total 2215 His CAU 594.00 0.3738 His CAC995.00 0.6262 Total 1589 Gln CAA 747.00 0.2783 Gln CAG 1937.00 0.7217Total 2684 Asn AAU 1109.00 0.3949 Asn AAC 1699.00 0.6051 Total 2808 LysAAA 1445.00 0.4088 Lys AAG 2090.00 0.5912 Total 3535 Asp GAU 1255.000.4055 Asp GAC 1840.00 0.5945 Total 3095 Glu GAA 1637.00 0.4164 Glu GAG2294.00 0.5836 Total 3931 Cys UGU 719.00 0.4425 Cys UGC 906.00 0.5575Total 1625 Trp UGG 1073.00 1.0000 Total 1073 Arg CGU 236.00 0.0700 ArgCGC 629.00 0.1865 Arg CGA 354.00 0.1050 Arg CGG 662.00 0.1963 Arg AGA712.00 0.2112 Arg AGG 779.00 0.2310 Total 3372 Gly GGU 648.00 0.1498 GlyGGC 1536.00 0.3551 Gly GGA 1065.00 0.2462 Gly GGG 1077.00 0.2490 Total4326 Stop UAA 55 Stop UAG 36 Stop UGA 110

TABLE 5 Codon Usage Table for Cow Genes (Bos taurus) Amino Acid CodonNumber Frequency of usage Phe UUU 13002 0.4112 Phe UUC 18614 0.5888Total 31616 Leu UUA 4467 0.0590 Leu UUG 9024 0.1192 Leu CUU 9069 0.1198Leu CUC 16003 0.2114 Leu CUA 4608 0.0609 Leu CUG 32536 0.4298 Total75707 Ile AUU 12474 0.3313 Ile AUC 19800 0.5258 Ile AUA 5381 0.1429Total 37655 Met AUG 17770 1.0000 Total 17770 Val GUU 8212 0.1635 Val GUC12846 0.2558 Val GUA 4932 0.0982 Val GUG 24222 0.4824 Total 50212 SerUCU 10287 0.1804 Ser UCC 13258 0.2325 Ser UCA 7678 0.1347 Ser UCG 34700.0609 Ser AGU 8040 0.1410 Ser AGC 14279 0.2505 Total 57012 Pro CCU11695 0.2684 Pro CCC 15221 0.3493 Pro CCA 11039 0.2533 Pro CCG 56210.1290 Total 43576 Thr ACU 9372 0.2203 Thr ACC 16574 0.3895 Thr ACA10892 0.2560 Thr ACG 5712 0.1342 Total 42550 Ala GCU 13923 0.2592 AlaGCC 23073 0.4295 Ala GCA 10704 0.1992 Ala GCG 6025 0.1121 Total 53725Tyr UAU 9441 0.3882 Tyr UAC 14882 0.6118 Total 24323 His CAU 6528 0.3649His CAC 11363 0.6351 Total 17891 Gln CAA 8060 0.2430 Gln CAG 251080.7570 Total 33168 Asn AAU 12491 0.4088 Asn AAC 18063 0.5912 Total 30554Lys AAA 17244 0.3897 Lys AAG 27000 0.6103 Total 44244 Asp GAU 166150.4239 Asp GAC 22580 0.5761 Total 39195 Glu GAA 21102 0.4007 Glu GAG31555 0.5993 Total 52657 Cys UGU 7556 0.4200 Cys UGC 10436 0.5800 Total17992 Trp UGG 10706 1.0000 Total 10706 Arg CGU 3391 0.0824 Arg CGC 79980.1943 Arg CGA 4558 0.1108 Arg CGG 8300 0.2017 Arg AGA 8237 0.2001 ArgAGG 8671 0.2107 Total 41155 Gly GGU 8508 0.1616 Gly GGC 18517 0.3518 GlyGGA 12838 0.2439 Gly GGG 12772 0.2427 Total 52635 Stop UAA 555 Stop UAG394 Stop UGA 392

By utilizing these or similar tables, one of ordinary skill in the artcan apply the frequencies to any given polypeptide sequence, and producea nucleic acid fragment of a codon-optimized coding region which encodesthe polypeptide, but which uses codons more optimal for a given species.Codon-optimized coding regions can be designed by various differentmethods.

In another method, termed “full-optimization,” the actual frequencies ofthe codons are distributed randomly throughout the coding region. Thus,using this method for optimization, if a hypothetical polypeptidesequence had 100 leucine residues, referring to Table 2 for frequency ofusage in humans, about 7, or 7% of the leucine codons would be UUA,about 13, or 13% of the leucine codons would be UUG, about 13, or 13% ofthe leucine codons would be CUU, about 20, or 20% of the leucine codonswould be CUC, about 7, or 7% of the leucine codons would be CUA, andabout 41, or 41% of the leucine codons would be CUG. These frequencieswould be distributed randomly throughout the leucine codons in thecoding region encoding the hypothetical polypeptide. As will beunderstood by those of ordinary skill in the art, the distribution ofcodons in the sequence can vary significantly using this method;however, the sequence always encodes the same polypeptide.

In using the “full-optimization” method, an entire polypeptide sequencemay be codon-optimized as described above. With respect to variousdesired fragments, variants or derivatives of the complete polypeptide,the fragment variant, or derivative may first be designed, and is thencodon-optimized individually. Alternatively, a full-length polypeptidesequence is codon-optimized for a given species resulting in acodon-optimized coding region encoding the entire polypeptide, and thennucleic acid fragments of the codon-optimized coding region, whichencode fragments, variants, and derivatives of the polypeptide are madefrom the original codon-optimized coding region. As would be wellunderstood by those of ordinary skill in the art, if codons have beenrandomly assigned to the full-length coding region based on theirfrequency of use in a given species, nucleic acid fragments encodingfragments, variants, and derivatives would not necessarily be fullycodon-optimized for the given species. However, such sequences are stillmuch closer to the codon usage of the desired species than the nativecodon usage. The advantage of this approach is that synthesizingcodon-optimized nucleic acid fragments encoding each fragment, variant,and derivative of a given polypeptide, although routine, would be timeconsuming and would result in significant expense.

When using the “full-optimization” method, the term “about” is usedprecisely to account for fractional percentages of codon frequencies fora given amino acid. As used herein, “about” is defined as one amino acidmore or one amino acid less than the value given. The whole number valueof amino acids is rounded up if the fractional frequency of usage is0.50 or greater, and is rounded down if the fractional frequency of useis 0.49 or less. Using again the example of the frequency of usage ofleucine in human genes for a hypothetical polypeptide having 62 leucineresidues, the fractional frequency of codon usage would be calculated bymultiplying 62 by the frequencies for the various codons. Thus, 7.28percent of 62 equals 4.51 UUA codons, or “about 5,” i.e., 4, 5, or 6 UUAcodons, 12.66 percent of 62 equals 7.85 UUG codons or “about 8,” i.e.,7, 8, or 9 TUG codons, 12.87 percent of 62 equals 7.98 CUU codons, or“about 8,” i.e., 7, 8, or 9 CTU codons, 19.56 percent of 62 equals 12.13CUC codons or “about 12,” i.e., 11, 12, or 13 CUC codons, 7.00 percentof 62 equals 4.34 CUA codons or “about 4,” i.e., 3, 4, or 5 CUA codons,and 40.62 percent of 62 equals 25.19 CUG codons, or “about 25,” i.e.,24, 25, or 26 CUG codons.

In a third method termed “minimal optimization,” coding regions are onlypartially optimized. For example, the invention includes a nucleic acidfragment of a codon-optimized coding region encoding a polypeptide inwhich at least about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% ofthe codon positions have been codon-optimized for a given species. Thatis, they contain a codon that is preferentially used in the genes of adesired species, e.g., a vertebrate species, e.g., humans, in place of acodon that is normally used in the native nucleic acid sequence. Codonsthat are rarely found in the genes of the vertebrate of interest arechanged to codons more commonly utilized in the coding regions of thevertebrate of interest.

This minimal human codon optimization for highly variant codons hasseveral advantages, which include but are not limited to the followingexamples. Since fewer changes are made to the nucleotide sequence of thegene of interest, fewer manipulations are required, which leads toreduced risk of introducing unwanted mutations and lower cost, as wellas allowing the use of commercially available site-directed mutagenesiskits, and reducing the need for expensive oligonucleotide synthesis.Further, decreasing the number of changes in the nucleotide sequencedecreases the potential of altering the secondary structure of thesequence, which can have a significant impact on gene expression incertain host cells. The introduction of undesirable restriction sites isalso reduced, facilitating the subcloning of the genes of interest intothe plasmid expression vector.

The present invention also provides isolated polynucleotides comprisingcoding regions of herpes simplex virus polypeptides, e.g., gD, VP 11/12,VP13/14 and/or VP22 or fragments, variants, or derivatives thereof. Theisolated polynucleotides can also be codon-optimized.

A human codon-optimized coding region can be designed by any of themethods discussed herein. For “uniform” optimization, each amino acid isassigned the most frequent codon used in the human genome for that aminoacid.

As described above, the term “about” means that the number of aminoacids encoded by a certain codon may be one more or one less than thenumber given. It would be understood by those of ordinary skill in theart that the total number of any amino acid in the polypeptide sequencemust remain constant, therefore, if there is one “more” of one codonencoding a give amino acid, there would have to be one “less” of anothercodon encoding that same amino acid.

In another form of minimal optimization, a Codon Usage Table (CUT) forthe specific herpes simplex virus sequence in question is generated andcompared to CUT for human genomic DNA. Amino acids are identified forwhich there is a difference of at least 10 percentage points in codonusage between human and herpes simplex virus DNA (either more or less).Then the wild type herpes simplex virus codon is modified to conform topredominant human codon for each such amino acid. Furthermore, theremainder of codons for that amino acid are also modified such that theyconform to the predominant human codon for each such amino acid.

Compositions and Methods

In certain embodiments, the present invention is directed tocompositions and methods of enhancing the immune response of avertebrate in need of protection against herpes simplex virus infectionby administering in vivo, into a tissue of a vertebrate, one or morepolynucleotides comprising at least one codon-optimized coding regionencoding a herpes simplex virus polypeptide, or a fragment, variant, orderivative thereof. In addition, the present invention is directed tocompositions and methods of enhancing the immune response of avertebrate in need of protection against herpes simplex virus infectionby administering to the vertebrate a composition comprising one or morepolynucleotides as described herein, and at least one isolated herpessimplex virus polypeptide, or a fragment, variant, or derivativethereof. The polynucleotide may be administered either prior to, at thesame time (simultaneously), or subsequent to the administration of theisolated polypeptide.

The coding regions encoding herpes simplex virus polypeptides orfragments, variants, or derivatives thereof may be codon optimized for aparticular vertebrate. Codon optimization is carried out by the methodsdescribed herein, for example, in certain embodiments codon-optimizedcoding regions encoding polypeptides of herpes simplex virus, or nucleicacid fragments of such coding regions encoding fragments, variants, orderivatives thereof are optimized according to the codon usage of theparticular vertebrate. The polynucleotides of the invention areincorporated into the cells of the vertebrate in vivo, and animmunologically effective amount of a herpes simplex virus polypeptideor a fragment, variant, or derivative thereof is produced in vivo. Thecoding regions encoding a herpes simplex virus polypeptide or afragment, variant, or derivative thereof may be codon optimized formammals, e.g., humans, apes, monkeys (e.g., owl, squirrel, cebus,rhesus, African green, patas, cynomolgus, and cercopithecus),orangutans, baboons, gibbons, and chimpanzees, dogs, wolves, cats,lions, and tigers, horses, donkeys, zebras, cows, pigs, sheep, deer,giraffes, bears, rabbits, mice, ferrets, seals, whales; birds, e.g.,ducks, geese, terns, shearwaters, gulls, turkeys, chickens, quail,pheasants, geese, starlings and budgerigars, or other vertebrates.

In one embodiment, the present invention relates to codon-optimizedcoding regions encoding polypeptides of herpes simplex virus, or nucleicacid fragments of such coding regions fragments, variants, orderivatives thereof which have been optimized according to human codonusage. For example, human codon-optimized coding regions encodingpolypeptides of herpes simplex virus, or fragments, variants, orderivatives thereof are prepared by substituting one or more codonspreferred for use in human genes for the codons naturally used in theDNA sequence encoding the herpes simplex virus polypeptide or afragment, variant, or derivative thereof. Also provided arepolynucleotides, vectors, and other expression constructs comprisingcodon-optimized coding regions encoding polypeptides of herpes simplexvirus, or nucleic acid fragments of such coding regions encodingfragments, variants, or derivatives thereof, pharmaceutical compositionscomprising polynucleotides, vectors, and other expression constructscomprising codon-optimized coding regions encoding polypeptides ofherpes simplex virus, or nucleic acid fragments of such coding regionsencoding fragments, variants, or derivatives thereof, and variousmethods of using such polynucleotides, vectors and other expressionconstructs. Coding regions encoding herpes simplex virus polypeptidescan be uniformly optimized, fully optimized, minimally optimized,codon-optimized by region and/or not codon-optimized, as describedherein.

The present invention is further directed towards polynucleotidescomprising codon-optimized coding regions encoding polypeptides ofherpes simplex virus antigens, for example, gD, VP 11/12, VP13/14 and/orVP22 optionally in conjunction with other antigens. The invention isalso directed to polynucleotides comprising codon-optimized nucleic acidfragments encoding fragments, variants and derivatives of thesepolypeptides.

In certain embodiments, the present invention provides an isolatedpolynucleotide comprising a nucleic acid fragment, where the nucleicacid fragment is a fragment of a codon-optimized coding region encodinga polypeptide at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to aherpes simplex virus polypeptide, e.g., gD, VP 11/12, VP13/14 and/orVP22 and where the nucleic acid fragment is a variant of acodon-optimized coding region encoding a herpes simplex viruspolypeptide, e.g., gD, VP 11/12, VP13/14 and/or VP22. The humancodon-optimized coding region can be optimized for any vertebratespecies and by any of the methods described herein.

Isolated Herpes Simplex Virus Polypeptides

The present invention is further drawn to compositions which include atleast one polynucleotide comprising one or more nucleic acid fragments,where each nucleic acid fragment is optionally a fragment of acodon-optimized coding region operably encoding a herpes simplex viruspolypeptide or fragment, variant, or derivative thereof together withone or more isolated herpes simplex virus component or isolatedpolypeptide. The herpes simplex virus component may be inactivatedvirus, attenuated virus, a viral vector expressing an isolated herpessimplex virus polypeptide, or a herpes simplex virus protein, fragment,variant or derivative thereof.

The isolated herpes simplex virus polypeptides of the invention may bein any form, and are generated using techniques well known in the art.Examples include isolated herpes simplex virus proteins producedrecombinantly, isolated herpes simplex virus proteins directly purifiedfrom their natural milieu, recombinant (non-herpes simplex virus) virusvectors expressing an isolated herpes simplex virus protein, or proteinsdelivered in the form of an inactivated herpes simplex virus vaccine,such as conventional vaccines.

In the instant invention, the combination of conventional antigenvaccine compositions with the codon-optimized nucleic acid compositionsprovides for therapeutically beneficial effects at dose sparingconcentrations. For example, immunological responses sufficient for atherapeutically beneficial effect in patients predetermined for anapproved commercial product, such as for the conventional productdescribed above, can be attained by using less of the approvedcommercial product when supplemented or enhanced with the appropriateamount of codon-optimized nucleic acid. Thus, dose sparing iscontemplated by administration of conventional herpes simplex virusvaccines administered in combination with the codon-optimized nucleicacids of the invention

In particular, the dose of conventional vaccine may be reduced by atleast 5%, at least 10%, at least 20%, at least 30%, at least 40%, atleast 50%, at least 60% or at least 70% when administered in combinationwith the codon-optimized nucleic acid compositions of the invention.

Similarly, a desirable level of an immunological response afforded by aDNA based pharmaceutical alone may be attained with less DNA byincluding an aliquot of a conventional vaccine. Further, using acombination of conventional and DNA based pharmaceuticals may allow bothmaterials to be used in lesser amounts while still affording the desiredlevel of immune response arising from administration of either componentalone in higher amounts (e.g. one may use less of either immunologicalproduct when they are used in combination). This may be manifest notonly by using lower amounts of materials being delivered at any time,but also to reducing the number of administrations points in avaccination regime (e.g. 2 versus 3 or 4 injections), and/or to reducingthe kinetics of the immunological response (e.g. desired response levelsare attained in 3 weeks instead of 6 after immunization).

In particular, the dose of DNA based pharmaceuticals, may be reduced byat least 5%, at least 10%, at least 20%, at least 30%, at least 40%, atleast 50%, at least 60% or at least 70% when administered in combinationwith conventional herpes simplex virus vaccines.

Determining the precise amounts of DNA based pharmaceutical andconventional antigen is based on a number of factors as described above,and is readily determined by one of ordinary skill in the art.

In addition to dose sparing, the claimed combinatorial compositionsprovide for a broadening of the immune response and/or enhancedbeneficial immune responses. Such broadened or enhanced immune responsesare achieved by: adding DNA to enhance cellular responses to aconventional vaccine; adding a conventional vaccine to a DNApharmaceutical to enhance humoral response; using a combination thatinduces additional epitopes (both humoral and/or cellular) to berecognized and/or more desirably responded to (epitope broadening);employing a DNA-conventional vaccine combination designed for aparticular desired spectrum of immunological responses; obtaining adesirable spectrum by using higher amounts of either component. Thebroadened immune response is measurable by one of ordinary skill in theart by standard immunological assay specific for the desirable responsespectrum.

Both broadening and dose sparing can be obtained simultaneously.

The isolated herpes simplex virus polypeptide or fragment, variant, orderivative thereof to be delivered (either a recombinant protein, apurified subunit, or viral vector expressing an isolated herpes simplexvirus polypeptide, or in the form of an inactivated herpes simplex virusvaccine) can be any isolated herpes simplex virus polypeptide orfragment, variant, or derivative thereof, including but not limited tothe gD, VP 11/12, VP13/14 and/or VP22 proteins or fragments, variants orderivatives thereof. It should be noted that any isolated herpes simplexvirus polypeptide or fragment, variant, or derivative thereof describedherein can be combined in a composition with any polynucleotidecomprising a nucleic acid fragment, where the nucleic acid fragment isoptionally a fragment of a codon-optimized coding region operablyencoding a herpes simplex virus polypeptide or fragment, variant, orderivative thereof. The proteins can be different, the same, or can becombined in any combination of one or more isolated herpes simplex virusproteins and one or more polynucleotides.

In certain embodiments, the isolated herpes simplex virus polypeptides,or fragments, derivatives or variants thereof can be fused to orconjugated to a second isolated herpes simplex virus polypeptide, orfragment, derivative or variant thereof, or can be fused to otherheterologous proteins, including for example, hepatitis B proteinsincluding, but not limited to the hepatitis B core antigen (HBcAg), orthose derived from diphtheria or tetanus. The second isolated herpessimplex virus polypeptide or other heterologous protein can act as a“carrier” that potentiates the immunogenicity of the herpes simplexvirus polypeptide or a fragment, variant, or derivative thereof to whichit is attached. Hepatitis B virus proteins and fragments and variantsthereof useful as carriers within the scope of the invention aredisclosed in U.S. Pat. Nos. 6,231,864 and 5,143,726. Polynucleotidescomprising coding regions encoding said fused or conjugated proteins arealso within the scope of the invention.

The use of recombinant particles comprising hepatitis B core antigen(“HBcAg”) and heterologous protein sequences as potent immunogenicmoieties is well documented. For example, addition of heterologoussequences to the amino terminus of a recombinant HBcAg results in thespontaneous assembly of particulate structures which express theheterologous epitope on their surface, and which are highly immunogenicwhen inoculated into experimental animals. See Clarke et al., Nature330:381-384 (1987). Heterologous epitopes can also be inserted intoHBcAg particles by replacing approximately 40 amino acids of the carboxyterminus of the protein with the heterologous sequences. Theserecombinant HBcAg proteins also spontaneously form immunogenicparticles. See Stahl and Murray, Proc. Natl. Acad. Sci. USA,86:6283-6287 (1989). Additionally, chimeric HBcAg particles may beconstructed where the heterologous epitope is inserted in or replacesall or part of the sequence of amino acid residues in a more centralregion of the HBcAg protein, in an immunodominant loop, thereby allowingthe heterologous epitope to be displayed on the surface of the resultingparticles. See EP Patent No. 0421635 B1 and Galibert, F., et al., Nature281:646-650 (1979); see also U.S. Pat. Nos. 4,818,527, 4,882,145 and5,143,726.

Chimaeric HBcAg particles comprising isolated herpes simplex virusproteins or variants, fragments or derivatives thereof are prepared byrecombinant techniques well known to those of ordinary skill in the art.A polynucleotide, e.g., a plasmid, which carries the coding region forthe HBcAg operably associated with a promoter is constructed. Convenientrestrictions sites are engineered into the coding region encoding theN-terminal, central, and/or C-terminal portions of the HBcAg, such thatheterologous sequences may be inserted. A construct which expresses aHBcAg/herpes simplex virus fusion protein is prepared by inserting a DNAsequence encoding a herpes simplex virus protein or variant, fragment orderivative thereof, in frame, into a desired restriction site in thecoding region of the HBcAg. The resulting construct is then insertedinto a suitable host cell, e.g., E. coli, under conditions where thechimeric HBcAg will be expressed. The chimaeric HBcAg self-assemblesinto particles when expressed, and can then be isolated, e.g., byultracentrifugation. The particles formed resemble the natural 27 nmHBcAg particles isolated from a hepatitis B virus, except that anisolated herpes simplex virus protein or fragment, variant, orderivative thereof is contained in the particle, preferably exposed onthe outer particle surface.

The herpes simplex virus protein or fragment, variant, or derivativethereof expressed in a chimaeric HBcAg particle may be of any size whichallows suitable particles of the chimeric HBcAg to self-assemble. Asdiscussed above, even small antigenic epitopes may be immunogenic whenexpressed in the context of an immunogenic carrier, e.g., a HBcAg. Thus,HBcAg particles of the invention may comprise at least 4, at least 5, atleast 6, at least 7, at least 8, at least 9, at least 10, at least 15,at least 20, at least 25, or between about 15 to about 30 amino acids ofa herpes simplex virus protein fragment of interest inserted therein.HBcAg particles of the invention may further comprise immunogenic orantigenic epitopes of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid residues of aherpes simplex virus protein fragment of interest inserted therein.

The immunodominant loop region of HBcAg was mapped to about amino acidresidues 75 to 83, to about amino acids 75 to 85 or to about amino acids130 to 140. See Colucci et al., J. Immunol. 141:4376-4380 (1988), andSalfeld et al. J. Virol. 63:798 (1989). A chimeric HBcAg is still oftenable to form core particles when foreign epitopes are cloned into theimmunodominant loop. Thus, for example, amino acids of the herpessimplex virus protein fragment may be inserted into the sequence ofHBcAg amino acids at various positions, for example, at the N-terminus,from about amino acid 75 to about amino acid 85, from about amino acid75 to about amino acid 83, from about amino acid 130 to about amino acid140, or at the C-terminus. Where amino acids of the herpes simplex virusprotein fragment replace all or part of the native core proteinsequence, the inserted herpes simplex virus sequence is generally notshorter, but may be longer, than the HBcAg sequence it replaces.

Alternatively, if particle formation is not desired, full-length herpessimplex virus coding sequences can be fused to the coding region for theHBcAg. The HBcAg sequences can be fused either at the N- or C-terminusof any of the Herpes simplex antigens described herein. Fusions couldinclude flexible protein linkers. These fusion constructs could be codonoptimized by any of the methods described.

The chimeric HBcAg can be used in the present invention in conjunctionwith a polynucleotide comprising a nucleic acid fragment, where eachnucleic acid fragment is optionally a fragment of a codon-optimizedcoding region operably encoding a herpes simplex virus polypeptide, or afragment, variant, or derivative thereof, as a herpes simplex vaccinefor a vertebrate.

Methods and Administration

The present invention also provides methods for delivering a herpessimplex virus polypeptide or a fragment, variant, or derivative thereofto a human, which comprise administering to a human one or more of thecompositions described herein; such that upon administration ofcompositions such as those described herein, a herpes simplex viruspolypeptide or a fragment, variant, or derivative thereof is expressedin human cells, in an amount sufficient to generate an immune responseto the herpes simplex virus or administering the herpes simplex viruspolypeptide or a fragment, variant, or derivative thereof itself to thehuman in an amount sufficient to generate an immune response.

The present invention further provides methods for delivering a herpessimplex virus polypeptide or a fragment, variant, or derivative thereofto a human, which comprise administering to a vertebrate one or more ofthe compositions described herein; such that upon administration ofcompositions such as those described herein, an immune response isgenerated in the vertebrate.

The term “vertebrate” is intended to encompass a singular “vertebrate”as well as plural “vertebrates” and comprises mammals and birds, as wellas fish, reptiles, and amphibians.

The term “mammal” is intended to encompass a singular “mammal” andplural “mammals,” and includes, but is not limited to humans; primatessuch as apes, monkeys (e.g., owl, squirrel, cebus, rhesus, Africangreen, patas, cynomolgus, and cercopithecus), orangutans, baboons,gibbons, and chimpanzees; canids such as dogs and wolves; felids such ascats, lions, and tigers; equines such as horses, donkeys, and zebras,food animals such as cows, pigs, and sheep; ungulates such as deer andgiraffes; ursids such as bears; and others such as rabbits, mice,ferrets, seals, whales. In particular, the mammal can be a humansubject, a food animal or a companion animal.

The term “bird” is intended to encompass a singular “bird” and plural“birds,” and includes, but is not limited to, feral water birds such asducks, geese, terns, shearwaters, and gulls; as well as domestic avianspecies such as turkeys, chickens, quail, pheasants, geese, and ducks.The term “bird” also encompasses passerine birds such as starlings andbudgerigars.

The present invention further provides a method for generating,enhancing or modulating an immune response to a herpes simplex viruscomprising administering to a vertebrate one or more of the compositionsdescribed herein. In this method, the compositions may include one ormore isolated polynucleotides comprising at least one nucleic acidfragment where the nucleic acid fragment is optionally a fragment of acodon-optimized coding region encoding a herpes simplex viruspolypeptide, or a fragment, variant, or derivative thereof. In anotherembodiment, the compositions may include both a polynucleotide asdescribed above, and also an isolated herpes simplex virus polypeptide,or a fragment, variant, or derivative thereof, wherein the protein isprovided as a recombinant protein, in particular, a fusion protein, apurified subunit, viral vector expressing the protein, or in the form ofan inactivated herpes simplex virus vaccine. Thus, the lattercompositions include both a polynucleotide encoding a herpes simplexvirus polypeptide or a fragment, variant, or derivative thereof and anisolated herpes simplex virus polypeptide or a fragment, variant, orderivative thereof. The herpes simplex virus polypeptide or a fragment,variant, or derivative thereof encoded by the polynucleotide of thecompositions need not be the same as the isolated herpes simplex viruspolypeptide or a fragment, variant, or derivative thereof of thecompositions. Compositions to be used according to this method may beunivalent, bivalent, trivalent or multivalent.

The polynucleotides of the compositions may comprise a fragment of ahuman (or other vertebrate) codon-optimized coding region encoding aprotein of the herpes simplex virus, or a fragment, variant, orderivative thereof. The polynucleotides are incorporated into the cellsof the vertebrate in vivo, and an antigenic amount of the herpes simplexvirus polypeptide, or fragment, variant, or derivative thereof, isproduced in vivo. Upon administration of the composition according tothis method, the herpes simplex virus polypeptide or a fragment,variant, or derivative thereof is expressed in the vertebrate in anamount sufficient to elicit an immune response. Such an immune responsemight be used, for example, to generate antibodies to the herpes simplexvirus for use in diagnostic assays or as laboratory reagents, or astherapeutic or preventative vaccines as described herein.

The present invention further provides a method for generating,enhancing, or modulating a protective and/or therapeutic immune responseto herpes simplex virus in a vertebrate, comprising administering to avertebrate in need of therapeutic and/or preventative immunity one ormore of the compositions described herein. In this method, thecompositions include one or more polynucleotides comprising at least onenucleic acid fragment, where the nucleic acid fragment is optionally afragment of a codon-optimized coding region encoding a herpes simplexvirus polypeptide, or a fragment, variant, or derivative thereof. In afurther embodiment, the composition used in this method includes both anisolated polynucleotide comprising at least one nucleic acid fragment,where the nucleic acid fragment is optionally a fragment of acodon-optimized coding region encoding a herpes simplex viruspolypeptide, or a fragment, variant, or derivative thereof; and at leastone isolated herpes simplex virus polypeptide, or a fragment, variant,or derivative thereof. Thus, the latter composition includes both anisolated polynucleotide encoding a herpes simplex virus polypeptide or afragment, variant, or derivative thereof and an isolated herpes simplexvirus polypeptide or a fragment, variant, or derivative thereof, forexample, a recombinant protein, a purified subunit, viral vectorexpressing the protein, or an inactivated virus vaccine. Uponadministration of the composition according to this method, the herpessimplex virus polypeptide or a fragment, variant, or derivative thereofis expressed in the human in a therapeutically or prophylacticallyeffective amount.

As used herein, an “immune response” refers to the ability of avertebrate to elicit an immune reaction to a composition delivered tothat vertebrate. Examples of immune responses include an antibodyresponse or a cellular, e.g., cytotoxic T-cell, response. One or morecompositions of the present invention may be used to prevent herpessimplex infection in vertebrates, e.g., as a prophylactic vaccine, toestablish or enhance immunity to herpes simplex virus in a healthyindividual prior to exposure to herpes simplex or contraction of herpessimplex disease, thus preventing the disease or reducing the severity ofdisease symptoms.

As mentioned above, compositions of the present invention can be usedboth to prevent herpes simplex virus infection, and also totherapeutically treat herpes simplex virus infection. In individualsalready exposed to herpes simplex, or already suffering from herpessimplex disease, the present invention is used to further stimulate theimmune system of the vertebrate, thus reducing or eliminating thesymptoms associated with that disease or disorder. As defined herein,“treatment” refers to the use of one or more compositions of the presentinvention to prevent, cure, retard, or reduce the severity of herpessimplex disease symptoms in a vertebrate, and/or result in no worseningof herpes simplex disease over a specified period of time in avertebrate which has already been exposed to herpes simplex virus and isthus in need of therapy. The term “prevention” refers to the use of oneor more compositions of the present invention to generate immunity in avertebrate which has not yet been exposed to a particular strain ofherpes simplex virus, thereby preventing or reducing disease symptoms ifthe vertebrate is later exposed to the particular strain of herpessimplex virus. The methods of the present invention therefore may bereferred to as therapeutic vaccination or preventative or prophylacticvaccination. It is not required that any composition of the presentinvention provide total immunity to herpes simplex or totally cure oreliminate all herpes simplex disease symptoms. As used herein, a“vertebrate in need of therapeutic and/or preventative immunity” refersto an individual for whom it is desirable to treat, i.e., to prevent,cure, retard, or reduce the severity of herpes simplex disease symptoms,and/or result in no worsening of herpes simplex disease over a specifiedperiod of time.

One or more compositions of the present invention are utilized in a“prime boost” regimen. An example of a “prime boost” regimen may befound in Yang, Z. et al. J. Virol. 77:799-803 (2002). In theseembodiments, one or more polynucleotide vaccine compositions of thepresent invention are delivered to a vertebrate, thereby priming theimmune response of the vertebrate to a herpes simplex virus, and then asecond immunogenic composition is utilized as a boost vaccination. Oneor more compositions of the present invention are used to primeimmunity, and then a second immunogenic composition, e.g., a recombinantviral vaccine or vaccines, a different polynucleotide vaccine, or one ormore purified subunit isolated herpes simplex virus polypeptides orfragments, variants or derivatives thereof is used to boost theanti-herpes simplex virus immune response.

In one embodiment, a priming composition and a boosting composition arecombined in a single composition or single formulation. For example, asingle composition may comprise an isolated herpes simplex viruspolypeptide or a fragment, variant, or derivative thereof as the primingcomponent and a polynucleotide encoding a herpes simplex protein as theboosting component. In this embodiment, the compositions may becontained in a single vial where the priming component and boostingcomponent are mixed together. In general, because the peak levels ofexpression of protein from the polynucleotide does not occur until later(e.g. 7-10 days) after administration, the polynucleotide component mayprovide a boost to the isolated protein component. Compositionscomprising both a priming component and a boosting component arereferred to herein as “combinatorial vaccine compositions” or “singleformulation heterologous prime-boost vaccine compositions.” In addition,the priming composition may be administered before the boostingcomposition, or even after the boosting composition, if the boostingcomposition is expected to take longer to act.

In another embodiment, the priming composition may be administeredsimultaneously with the boosting composition, but in separateformulations where the priming component and the boosting component areseparated.

The terms “priming” or “primary” and “boost” or “boosting” as usedherein may refer to the initial and subsequent immunizations,respectively, i.e., in accordance with the definitions these termsnormally have in immunology. However, in certain embodiments, e.g.,where the priming component and boosting component are in a singleformulation, initial and subsequent immunizations may not be necessaryas both the “prime” and the “boost” compositions are administeredsimultaneously.

In certain embodiments, one or more compositions of the presentinvention are delivered to a vertebrate by methods described herein,thereby achieving an effective therapeutic and/or an effectivepreventative immune response. More specifically, the compositions of thepresent invention may be administered to any tissue of a vertebrate,including, but not limited to, muscle, skin, brain tissue, lung tissue,liver tissue, spleen tissue, bone marrow tissue, thymus tissue, hearttissue, e.g., myocardium, endocardium, and pericardium, lymph tissue,blood tissue, bone tissue, pancreas tissue, kidney tissue, gall bladdertissue, stomach tissue, intestinal tissue, testicular tissue, ovariantissue, uterine tissue, vaginal tissue, rectal tissue, nervous systemtissue, eye tissue, glandular tissue, tongue tissue, and connectivetissue, e.g., cartilage.

Furthermore, the compositions of the present invention may beadministered to any internal cavity of a vertebrate, including, but notlimited to, the lungs, the mouth, the nasal cavity, the stomach, theperitoneal cavity, the intestine, any heart chamber, veins, arteries,capillaries, lymphatic cavities, the uterine cavity, the vaginal cavity,the rectal cavity, joint cavities, ventricles in brain, spinal canal inspinal cord, the ocular cavities, the lumen of a duct of a salivarygland or a liver. When the compositions of the present invention isadministered to the lumen of a duct of a salivary gland or liver, thedesired polypeptide is expressed in the salivary gland and the liversuch that the polypeptide is delivered into the blood stream of thevertebrate from each of the salivary gland or the liver. Certain modesfor administration to secretory organs of a gastrointestinal systemusing the salivary gland, liver and pancreas to release a desiredpolypeptide into the bloodstream is disclosed in U.S. Pat. Nos.5,837,693 and 6,004,944.

In certain embodiments, the compositions are administered to muscle,either skeletal muscle or cardiac muscle, or to lung tissue. Specific,but non-limiting modes for administration to lung tissue are disclosedin Wheeler, C. J., et al., Proc. Natl. Acad. Sci. USA 93:11454-11459(1996), which is incorporated herein by reference in its entirety.

According to the disclosed methods, compositions of the presentinvention can be administered by intramuscular (i.m.), interdermal(i.d.), subcutaneous (s.c), or intrapulmonary routes. Other suitableroutes of administration include, but are not limited to intratracheal,transdermal, intraocular, intranasal, inhalation, intracavity,intravenous (i.v.), intraductal (e.g., into the pancreas) andintraparenchymal (i.e., into any tissue) administration. Transdermaldelivery includes, but not limited to intradermal (e.g., into the dermisor epidermis), transdermal (e.g., percutaneous) and transmucosaladministration (i.e., into or through skin or mucosal tissue).Intracavity administration includes, but not limited to administrationinto oral, vaginal, rectal, nasal, peritoneal, or intestinal cavities aswell as, intrathecal (i.e., into spinal canal), intraventricular (i.e.,into the brain ventricles or the heart ventricles), intraatrial (i.e.,into the heart atrium) and sub arachnoid (i.e., into the sub arachnoidspaces of the brain) administration.

For oral indications, the present invention may be administered in theform of tongue strips wherein the composition is embedded or applied tothe strip. The user places the strip on the tongue and the strip meltsor dissolves in the mouth thereby releasing the composition.

Any mode of administration can be used so long as the mode results inthe expression of the desired peptide or protein, in the desired tissue,in an amount sufficient to generate an immune response to herpes simplexvirus and/or to generate a prophylactically or therapeutically effectiveimmune response to herpes simplex virus in a human in need of suchresponse. Administration means of the present invention include needleinjection, catheter infusion, biolistic injectors, particle accelerators(e.g., “gene guns” or pneumatic “needleless” injectors) Med-E-Jet(Vahlsing, H., et al., J. Immunol. Methods 171:11-22 (1994)), Pigjet(Schrijver, R., et al., Vaccine 15: 1908-1916 (1997)), Biojector (Davis,H., et al., Vaccine 12: 1503-1509 (1994); Gramzinski, R., et al., Mol.Med. 4: 109-118 (1998)), AdvantaJet (Linmayer, I., et al., Diabetes Care9:294-297 (1986)), Medi-jector (Martins, J., and Roedl, E. J. Occup.Med. 21:821-824 (1979)), U.S. Pat. No. 5,399,163; U.S. Pat. No.5,383,851; gelfoam sponge depots, other commercially available depotmaterials (e.g., hydrogels), osmotic pumps (e.g., Alza minipumps), oralor suppositorial solid (tablet or pill) pharmaceutical formulations,topical skin creams, and decanting, use of polynucleotide coated suture(Qin, Y., et al., Life Sciences 65:2193-2203 (1999)) or topicalapplications during surgery. Certain modes of administration areintramuscular or intradermal needle-based injection and pulmonaryapplication via catheter infusion. Energy-assisted plasmid delivery(EAPD) methods may also be employed to administer the compositions ofthe invention. One such method involves the application of briefelectrical pulses to injected tissues, a procedure commonly known aselectroporation. See generally Mir, L. M. et al., Proc. Natl. Acad. Sci.USA 96:4262-7 (1999); Hartikka, J. et al., Mol. Ther. 4:407-15 (2001);Mathiesen, I., Gene Ther. 6:508-14 (1999); Rizzuto G. et al., Hum. Gen.Ther. 11:1891-900 (2000).

Determining an effective amount of one or more compositions of thepresent invention depends upon a number of factors including, forexample, the antigen being expressed or administered directly, e.g., gD,VP 11/12, VP13/14 and/or VP22, or fragments, variants, or derivativesthereof, the age and weight of the subject, the precise conditionrequiring treatment and its severity, and the route of administration.Based on the above factors, determining the precise amount, number ofdoses, and timing of doses are within the ordinary skill in the art andwill be readily determined by the attending physician or veterinarian.

Compositions of the present invention may include various salts,excipients, delivery vehicles and/or auxiliary agents as are disclosed,e.g., in U.S. patent application Publication No. 2002/0019358, publishedFeb. 14, 2002.

Furthermore, compositions of the present invention may include one ormore transfection facilitating compounds that facilitate delivery ofpolynucleotides to the interior of a cell, and/or to a desired locationwithin a cell. As used herein, the terms “transfection facilitatingcompound,” “transfection facilitating agent,” and “transfectionfacilitating material” are synonymous, and may be used interchangeably.It should be noted that certain transfection facilitating compounds mayalso be “adjuvants” as described infra, i.e., in addition tofacilitating delivery of polynucleotides to the interior of a cell, thecompound acts to alter or increase the immune response to the antigenencoded by that polynucleotide. Examples of the transfectionfacilitating compounds include, but are not limited to, inorganicmaterials such as calcium phosphate, alum (aluminum sulfate), and goldparticles (e.g., “powder” type delivery vehicles); peptides that are,for example, canonic, intercell targeting (for selective delivery tocertain cell types), intracell targeting (for nuclear localization orendosomal escape), and ampipathic (helix forming or pore forming);proteins that are, for example, basic (e.g., positively charged) such ashistories, targeting (e.g., asialoprotein), viral (e.g., Sendai viruscoat protein), and pore-forming; lipids that are, for example, cationic(e.g., DMRIE, DOSPA, DC-Chol), basic (e.g., steryl amine), neutral(e.g., cholesterol), anionic (e.g., phosphatidyl serine), andzwitterionic (e.g., DOPE, DOPC); and polymers such as dendrimers,star-polymers, “homogenous” poly-amino acids (e.g., poly-lysine,poly-arginine), “heterogeneous” poly-amino acids (e.g., mixtures oflysine & glycine), co-polymers, polyvinylpyrrolidinone (PVP), poloxamers(e.g. CRL 1005) and polyethylene glycol (PEG). A transfectionfacilitating material can be used alone or in combination with one ormore other transfection facilitating materials. Two or more transfectionfacilitating materials can be combined by chemical bonding (e.g.,covalent and ionic such as in lipidated polylysine, PEGylatedpolylysine) (Toncheva, et al., Biochim. Biophys. Acta 1380(3):354-368(1988)), mechanical mixing (e.g., tree moving materials in liquid orsolid phase such as “polylysine+cationic lipids”) (Gao and Huang,Biochemistry 35:1027-1036 (1996); Trubetskoy, et al., Biochem. Biophys.Acta 1131:311-313 (1992)), and aggregation (e.g., co-precipitation, gelforming such as in cationic lipids+poly-lactide, andpolylysine+gelatin).

One category of transfection facilitating materials is cationic lipids.Examples of cationic lipids are 5-carboxyspermylglycine dioctadecylamide(DOGS) and dipalmitoyl-phophatidylethanolamine-5-carboxyspermylamide(DPPES). Cationic cholesterol derivatives are also useful, including{3β-[N—N′,N′-dimethylamino)ethane]-carbomoyl}-cholesterol (DC-Chol).Dimethyldioctdecyl-ammonium bromide (DDAB),N-(3-aminopropyl)-N,N-(bis-(2-tetradecyloxyethyl))-N-methyl-ammoniumbromide (PA-DEMO),N-(3-aminopropyl)-N,N-(bis-(2-dodecyloxyethyl))-N-methyl-ammoniumbromide (PA-DELO),N,N,N-tris-(2-dodecyloxy)ethyl-N-(3-amino)propyl-ammonium bromide(PA-TELO), andN1-(3-aminopropyl)((2-dodecyloxy)ethyl)-N2-(2-dodecyloxy)ethyl-1-piperazinaminiumbromide (GA-LOE-BP) can also be employed in the present invention.

Non-diether cationic lipids, such asDL-1,2-doleoyl-3-dimethylaminopropyl-β-hydroxyethylammonium (DORIdiester),1-O-oleyl-2-oleoyl-3-dimethylaminopropyl-p-hydroxyethylammonium (DORIester/ether), and their salts promote in vivo gene delivery. In someembodiments, cationic lipids comprise groups attached via a heteroatomattached to the quaternary ammonium moiety in the head group. A glycylspacer can connect the linker to the hydroxyl group.

Specific, but non-limiting cationic lipids for use in certainembodiments of the present invention include DMRIE((±)-N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminiumbromide), GAP-DMORIE((±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(syn-9-tetradeceneyloxy)-1-propanaminiumbromide), and GAP-DMRIE((±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-(bis-dodecyloxy)-1-propaniminiumbromide).

Other specific but non-limiting cationic surfactants for use in certainembodiments of the present invention include Bn-DHRIE, DhxRIE,DhxRIE-OAc, DhxRIE-OBz and Pr-DOctRIE-OAc. These lipids are disclosed incopending U.S. patent application Ser. No. 10/725,015. In another aspectof the present invention, the cationic surfactant is Pr-DOctRIE-OAc.

Other cationic lipids include(±)-N,N-dimethyl-N-[2-(sperminecarboxamido)ethyl]-2,3-bis(dioleyloxy)-1-propaniminiumpentahydrochloride (DOSPA),(±)—N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propaniminiumbromide (β-aminoethyl-DMRIE or βAE-DMRIE) (Wheeler, et al., Biochim.Biophys. Acta 1280:1-11 (1996), and(±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propaniminiumbromide (GAP-DLRIE) (Wheeler, et al., Proc. Natl. Acad. Sci. USA93:11454-11459 (1996)), which have been developed from DMRIE.

Other examples of DMRIE-derived cationic lipids that are useful for thepresent invention are(±)—N-(3-aminopropyl)-N,N-dimethyl-2,3-(bis-decyloxy)-1-propanaminiumbromide (GAP-DDRIE),(±)—N-(3-aminopropyl)-N,N-dimethyl-2,3-(bis-tetradecyloxy)-1-propanaminiumbromide (GAP-DMRIE),(±)—N—((N″-methyl)-N′-ureyl)propyl-N,N-dimethyl-2,3-bis(tetradecyloxy-)-1-propanaminiumbromide (GMU-DMRIE),(±)—N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanaminiumbromide (DLRIE), and(±)—N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis-([Z]-9-octadecenyloxy)propyl-1-propaniminiumbromide (HP-DORIE).

In the embodiments where the immunogenic composition comprises acationic lipid, the cationic lipid may be mixed with one or moreco-lipids. For purposes of definition, the term “co-lipid” refers to anyhydrophobic material which may be combined with the cationic lipidcomponent and includes amphipathic lipids, such as phospholipids, andneutral lipids, such as cholesterol. Cationic lipids and co-lipids maybe mixed or combined in a number of ways to produce a variety ofnon-covalently bonded macroscopic structures, including, for example,liposomes, multilamellar vesicles, unilamellar vesicles, micelles, andsimple films. One non-limiting class of co-lipids are the zwitterionicphospholipids, which include the phosphatidylethanolamines and thephosphatidylcholines. Examples of phosphatidylethanolamines, includeDOPE, DMPE and DPyPE. In certain embodiments, the co-lipid is DPyPEwhich comprises two phytanoyl substituents incorporated into thediacylphosphatidylethanolamine skeleton and the cationic lipid isGAP-DMORIE, (resulting in VAXFECTIN adjuvant). In other embodiments, theco-lipid is DOPE, the CAS name is1,2-diolyeoyl-sn-glycero-3-phosphoethanolamine.

When a composition of the present invention comprises a cationic lipidand co-lipid, the cationic lipid:co-lipid molar ratio may be from about9:1 to about 1:9, from about 4:1 to about 1:4, from about 2:1 to about1:2, or about 1:1.

In order to maximize homogeneity, the cationic lipid and co-lipidcomponents may be dissolved in a solvent such as chloroform, followed byevaporation of the cationic lipid/co-lipid solution under vacuum todryness as a film on the inner surface of a glass vessel (e.g., aRotovap round-bottomed flask). Upon suspension in an aqueous solvent,the amphipathic lipid component molecules self-assemble into homogenouslipid vesicles. These lipid vesicles may subsequently be processed tohave a selected mean diameter of uniform size prior to complexing with,for example, a codon-optimized polynucleotide of the present invention,according to methods known to those skilled in the art. For example, thesonication of a lipid solution is described in Feigner et al., Proc.Natl. Acad. Sci. USA 8: 7413-7417 (1987) and in U.S. Pat. No. 5,264,618.

In those embodiments where the composition includes a cationic lipid,polynucleotides of the present invention are complexed with lipids bymixing, for example, a plasmid in aqueous solution and a solution ofcationic lipid:co-lipid as prepared herein are mixed. The concentrationof each of the constituent solutions can be adjusted prior to mixingsuch that the desired final plasmid/cationic lipid:co-lipid ratio andthe desired plasmid final concentration will be obtained upon mixing thetwo solutions. The cationic lipid:co-lipid mixtures are suitablyprepared by hydrating a thin film of the mixed lipid materials in anappropriate volume of aqueous solvent by vortex mixing at ambienttemperatures for about 1 minute. The thin films are prepared by admixingchloroform solutions of the individual components to afford a desiredmolar solute ratio followed by aliquoting the desired volume of thesolutions into a suitable container. The solvent is removed byevaporation, first with a stream of dry, inert gas (e.g. argon) followedby high vacuum treatment.

Other hydrophobic and amphiphilic additives, such as, for example,sterols, fatty acids, gangliosides, glycolipids, lipopeptides,liposaccharides, neobees, niosomes, prostaglandins and sphingolipids,may also be included in compositions of the present invention. In suchcompositions, these additives may be included in an amount between about0.1 mol % and about 99.9 mol % (relative to total lipid), about 1-50 mol%, or about 2-25 mol %.

Additional embodiments of the present invention are drawn tocompositions comprising an auxiliary agent which is administered before,after, or concurrently with the polynucleotide. As used herein, an“auxiliary agent” is a substance included in a composition for itsability to enhance, relative to a composition which is identical exceptfor the inclusion of the auxiliary agent, the entry of polynucleotidesinto vertebrate cells in vivo, and/or the in vivo expression ofpolypeptides encoded by such polynucleotides. Certain auxiliary agentsmay, in addition to enhancing entry of polynucleotides into cells,enhance an immune response to an immunogen encoded by thepolynucleotide. Auxiliary agents of the present invention includenonionic, anionic, canonic, or zwitterionic surfactants or detergents,with nonionic surfactants or detergents being preferred, chelators,DNase inhibitors, poloxamers, agents that aggregate or condense nucleicacids, emulsifying or solubilizing agents, wetting agents, gel-formingagents, and buffers.

Auxiliary agents for use in compositions of the present inventioninclude, but are not limited to non-ionic detergents and surfactantsIGEPAL CA 6300 octylphenyl-polyethylene glycol, NONIDET NP-40nonylphenoxypolyethoxyethanol, NONIDET P-40octylphenoxypolyethoxyethanol, TWEEN-20 polysorbate 20, TWEEN-80polysorbate 80, PLURONIC F68 poloxamer (ave. MW: 8400; approx. MW ofhydrophobe, 1800; approx. wt. % of hydrophile, 80%), PLURONICF77poloxamer (ave. MW: 6600; approx. MW of hydrophobe, 2100; approx. wt.% of hydrophile, 70%), PLURONIC P65poloxamer (ave. MW: 3400; approx. MWof hydrophobe, 1800; approx. wt. % of hydrophile, 50%), TRITON X-1004-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol, and TRITON X-114(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol; the anionicdetergent sodium dodecyl sulfate (SDS); the sugar stachyose; thecondensing agent DMSO; and the chelator/DNAse inhibitor EDTA, CRL 1005(12 kpa, 5% POE), and BAK (Benzalkonium chloride 50% solution, availablefrom Ruger Chemical Co. Inc.). In certain specific embodiments, theauxiliary agent is DMSO, NONIDET P-40 octylphenoxypolyethoxyethanol,PLURONIC F68poloxamer (ave. MW: 8400; approx. MW of hydrophobe, 1800;approx. wt. % of hydrophile, 80%), PLURONIC F77poloxamer (ave. MW: 6600;approx. MW of hydrophobe, 2100; approx. wt. % of hydrophile, 70%),PLURONIC P65 (ave. MW: 3400; approx. MW of hydrophobe, 1800; approx. wt.% of hydrophile, 50%), PLURONIC L64poloxamer (ave. MW: 2900; approx. MWof hydrophobe, 1800; approx. wt. % of hydrophile, 40%), and PLURONICF108poloxamer (ave. MW: 14600; approx. MW of hydrophobe, 3000; approx.wt. % of hydrophile, 80%). See, e.g., U.S. patent applicationPublication No. 2002/0019358, published Feb. 14, 2002.

Certain compositions of the present invention can further include one ormore adjuvants before, after, or concurrently with the polynucleotide.The term “adjuvant” refers to any material having the ability to (1)alter or increase the immune response to a particular antigen or (2)increase or aid an effect of a pharmacological agent. It should benoted, with respect to polynucleotide vaccines, that an “adjuvant,” canbe a transfection facilitating material. Similarly, certain“transfection facilitating materials” described supra, may also be an“adjuvant.” An adjuvant maybe used with a composition comprising apolynucleotide of the present invention. In a prime-boost regimen, asdescribed herein, an adjuvant may be used with either the primingimmunization, the booster immunization, or both. Suitable adjuvantsinclude, but are not limited to, cytokines and growth factors; bacterialcomponents (e.g., endotoxins, in particular superantigens, exotoxins andcell wall components); aluminum-based salts; calcium-based salts;silica; polynucleotides; toxoids; serum proteins, viruses andvirally-derived materials, poisons, venoms, imidazoquiniline compounds,poloxamers, and cationic lipids.

A great variety of materials have been shown to have adjuvant activitythrough a variety of mechanisms. Any compound which may increase theexpression, antigenicity or immunogenicity of the polypeptide is apotential adjuvant. The present invention provides an assay to screenfor improved immune responses to potential adjuvants. Potentialadjuvants which may be screened for their ability to enhance the immuneresponse according to the present invention include, but are not limitedto: inert carriers, such as alum, bentonite, latex, and acrylicparticles; PLURONIC block polymers, such as TITERMAX (block copolymerCRL-8941, squalene (a metabolizable oil) and a microparticulate silicastabilizer); depot formers, such as Freunds adjuvant, surface activematerials, such as saponin, lysolecithin, retinal, Quil A, liposomes,and PLURONIC polymer formulations; macrophage stimulators, such asbacterial lipopolysaccharide; alternate pathway complement activators,such as insulin, zymosan, endotoxin, and levamisole; and non-ionicsurfactants, such as poloxamers, poly(oxyethylene)-poly(oxypropylene)tri-block copolymers. Also included as adjuvants aretransfection-facilitating materials, such as those described above.

Poloxamers which may be screened for their ability to enhance the immuneresponse according to the present invention include, but are not limitedto, commercially available poloxamers such as PLURONIC surfactants,which are block copolymers of propylene oxide and ethylene oxide inwhich the propylene oxide block is sandwiched between two ethylene oxideblocks. Examples of PLURONIC surfactants include PLURONIC L121 poloxamer(ave. MW: 4400; approx. MW of hydrophobe, 3600; approx. wt % ofhydrophile, 10%), PLURONIC L101 poloxamer (ave. MW: 3800; approx. MW ofhydrophobe, 3000; approx. wt. % of hydrophile, 10%), PLURONIC L81poloxamer (ave. MW: 2750; approx. MW of hydrophobe, 2400; approx. wt. %of hydrophile, 10%), PLURONIC L61 poloxamer (ave. MW: 2000; approx. MWof hydrophobe, 1800; approx. wt. % of hydrophile, 10%), PLURONIC L31poloxamer (ave. MW: 1100; approx. MW of hydrophobe, 900; approx. wt. %of hydrophile, 10%), PLURONIC L122 poloxamer (ave. MW: 5000; approx. MWof hydrophobe, 3600; approx. wt. % of hydrophile, 20%), PLURONIC L92poloxamer (ave. MW: 3650; approx. MW of hydrophobe, 2700; approx. wt. %of hydrophile, 20%), PLURONIC L72 poloxamer (ave. MW: 2750; approx. MWof hydrophobe, 2100; approx. wt. % of hydrophile, 20%), PLURONIC L62poloxamer (ave. MW: 2500; approx. MW of hydrophobe, 1800; approx. wt. %of hydrophile, 20%), PLURONIC L42 poloxamer (ave. MW: 1630; approx. MWof hydrophobe, 1200; approx. wt. % of hydrophile, 20%), PLURONIC L63poloxamer (ave. MW: 2650; approx. MW of hydrophobe, 1800; approx. wt. %of hydrophile, 30%), PLURONIC L43 poloxamer (ave. MW: 1850; approx. MWof hydrophobe, 1200; approx. wt. % of hydrophile, 30%), PLURONIC L64poloxamer (ave. MW: 2900; approx. MW of hydrophobe, 1800; approx. wt. %of hydrophile, 40%), PLURONIC L44 poloxamer (ave. MW: 2200; approx. MWof hydrophobe, 1200; approx. wt. % of hydrophile, 40%), PLURONIC L35poloxamer (ave. MW: 1900; approx. MW of hydrophobe, 900; approx. wt. %of hydrophile, 50%), PLURONIC P123 poloxamer (ave. MW: 5750; approx. MWof hydrophobe, 3600; approx. wt. % of hydrophile, 30%), PLURONIC P103poloxamer (ave. MW: 4950; approx. MW of hydrophobe, 3000; approx. wt. %of hydrophile, 30%), PLURONIC P104 poloxamer (ave. MW: 5900; approx. MWof hydrophobe, 3000; approx. wt. % of hydrophile, 40%), PLURONIC P84poloxamer (ave. MW: 4200; approx. MW of hydrophobe, 2400; approx. wt. %of hydrophile, 40%), PLURONIC P105 poloxamer (ave. MW: 6500; approx. MWof hydrophobe, 3000; approx. wt. % of hydrophile, 50%), PLURONIC P85poloxamer (ave. MW: 4600; approx. MW of hydrophobe, 2400; approx. wt. %of hydrophile, 50%), PLURONIC P75 poloxamer (ave. MW: 4150; approx. MWof hydrophobe, 2100; approx. wt. % of hydrophile, 50%), PLURONIC P65poloxamer (ave. MW: 3400; approx. MW of hydrophobe, 1800; approx. wt. %of hydrophile, 50%), PLURONIC F127 poloxamer (ave. MW: 12600; approx. MWof hydrophobe, 3600; approx. wt. % of hydrophile, 70%), PLURONIC F98poloxamer (ave. MW: 13000; approx. MW of hydrophobe, 2700; approx. wt. %of hydrophile, 80%), PLURONIC F87 poloxamer (ave. MW: 7700; approx. MWof hydrophobe, 2400; approx. wt. % of hydrophile, 70%), PLURONIC F77poloxamer (ave. MW: 6600; approx. MW of hydrophobe, 2100; approx. wt. %of hydrophile, 70%), PLURONIC F108 poloxamer (ave. MW: 14600; approx. MWof hydrophobe, 3000; approx. wt. % of hydrophile, 80%), PLURONIC F98poloxamer (ave. MW: 13000; approx. MW of hydrophobe, 2700; approx. wt. %of hydrophile, 80%), PLURONIC F88 poloxamer (ave. MW: 11400; approx. MWof hydrophobe, 2400; approx. wt. % of hydrophile, 80%), PLURONIC F68poloxamer (ave. MW: 8400; approx. MW of hydrophobe, 1800; approx. wt. %of hydrophile, 80%), PLURONIC F38 poloxamer (ave. MW: 4700; approx. MWof hydrophobe, 900; approx. wt. % of hydrophile, 80%).

Reverse poloxamers which may be screened for their ability to enhancethe immune response according to the present invention include, but arenot limited to PLURONIC R 31R1 reverse poloxamer (ave. MW: 3250; approx.MW of hydrophobe, 3100; approx. wt. % of hydrophile, 10%), PLURONICR25R1 reverse poloxamer (ave. MW: 2700; approx. MW of hydrophobe, 2500;approx. wt. % of hydrophile, 10%), PLURONIC R 17R1 reverse poloxamer(ave. MW: 1900; approx. MW of hydrophobe, 1700; approx. wt. % ofhydrophile, 10%), PLURONIC R 31R2 reverse poloxamer (ave. MW: 3300;approx. MW of hydrophobe, 3100; approx. wt. % of hydrophile, 20%),PLURONIC R 25R2 reverse poloxamer (ave. MW: 3100; approx. MW ofhydrophobe, 2500; approx. wt. % of hydrophile, 20%), PLURONIC R 17R2reverse poloxamer (ave. MW: 2150; approx. MW of hydrophobe, 1700;approx. wt. % of hydrophile, 20%), PLURONIC R 12R3 reverse poloxamer(ave. MW: 1800; approx. MW of hydrophobe, 1200; approx. wt. % ofhydrophile, 30%), PLURONIC R 31R4 reverse poloxamer (ave. MW: 4150;approx. MW of hydrophobe, 3100; approx. wt. % of hydrophile, 40%),PLURONIC R 25R4 reverse poloxamer (ave. MW: 3600; approx. MW ofhydrophobe, 2500; approx. wt. % of hydrophile, 40%), PLURONIC R 22R4reverse poloxamer (ave. MW: 3350; approx. MW of hydrophobe, 2200;approx. wt. % of hydrophile, 40%), PLURONIC R17R4 reverse poloxamer(ave. MW: 3650; approx. MW of hydrophobe, 1700; approx. wt. % ofhydrophile, 40%), PLURONIC R 25R5 reverse poloxamer (ave. MW: 4320;approx. MW of hydrophobe, 2500; approx. wt. % of hydrophile, 50%),PLURONIC R10R5 reverse poloxamer (ave. MW: 1950; approx. MW ofhydrophobe, 1000; approx. wt. % of hydrophile, 50%), PLURONIC R 25R8reverse poloxamer (ave. MW: 8550; approx. MW of hydrophobe, 2500;approx. wt. % of hydrophile, 80%), PLURONIC R 17R8 reverse poloxamer(ave. MW: 7000; approx. MW of hydrophobe, 1700; approx. wt. % ofhydrophile, 80%), and PLURONIC R 10R8 reverse poloxamer (ave. MW: 4550;approx. MW of hydrophobe, 1000; approx. wt. % of hydrophile, 80%).

Other commercially available poloxamers which may be screened for theirability to enhance the immune response according to the presentinvention include compounds that are block copolymer of polyethylene andpolypropylene glycol such as SYNPERONIC L121 (ave. MW: 4400), SYNPERONICL122 (ave. MW: 5000), SYNPERONIC P104 (ave. MW: 5850), SYNPERONIC P105(ave. MW: 6500), SYNPERONIC P123 (ave. MW: 5750), SYNPERONIC P85 (ave.MW: 4600) and SYNPERONIC P94 (ave. MW: 4600), in which L indicates thatthe surfactants are liquids, P that they are pastes, the first digit isa measure of the molecular weight of the polypropylene portion of thesurfactant and the last digit of the number, multiplied by 10, gives thepercent ethylene oxide content of the surfactant; and compounds that arenonylphenyl polyethylene glycol such as SYNPERONIC NP10 (nonylphenolethoxylated surfactant—10% solution), SYNPERONIC NP30 (condensate of 1mole of nonylphenol with 30 moles of ethylene oxide) and SYNPERONIC NP5(condensate of 1 mole of nonylphenol with 5.5 moles of naphthaleneoxide).

Other poloxamers which may be screened for their ability to enhance theimmune response according to the present invention include: (a) apolyether block copolymer comprising an A-type segment and a B-typesegment, wherein the A-type segment comprises a linear polymeric segmentof relatively hydrophilic character, the repeating units of whichcontribute an average Hansch-Leo fragmental constant of about −0.4 orless and have molecular weight contributions between about 30 and about500, wherein the B-type segment comprises a linear polymeric segment ofrelatively hydrophobic character, the repeating units of whichcontribute an average Hansch-Leo fragmental constant of about −0.4 ormore and have molecular weight contributions between about 30 and about500, wherein at least about 80% of the linkages joining the repeatingunits for each of the polymeric segments comprise an ether linkage; (b)a block copolymer having a polyether segment and a polycation segment,wherein the polyether segment comprises at least an A-type block, andthe polycation segment comprises a plurality of cationic repeatingunits; and (c) a polyether-polycation copolymer comprising a polymer, apolyether segment and a polycationic segment comprising a plurality ofcationic repeating units of formula —NH—R⁰, wherein R⁰ is a straightchain aliphatic group of 2 to 6 carbon atoms, which may be substituted,wherein said polyether segments comprise at least one of an A-type ofB-type segment. See U.S. Pat. No. 5,656,611. Other poloxamers ofinterest include CRL1005 (12 kDa, 5% POE), CRL8300 (11 kDa, 5% POE),CRL2690 (12 kDa, 10% POE), CRL4505 (15 kDa, 5% POE) and CRL1415 (9 kDa,10% POE).

Other auxiliary agents which may be screened for their ability toenhance the immune response according to the present invention include,but are not limited to, Acacia (gum arabic); the poloxyethylene etherR—O—(C₂H₄O)_(x)—H (BRIJ), e.g., polyethylene glycol dodecyl ether (BRIJ35, x=23), polyethylene glycol dodecyl ether (BRIJ 30, x=4),polyethylene glycol hexadecyl ether (BRIJ 52 x=2), polyethylene glycolhexadecyl ether (BRIJ 56, x=10), polyethylene glycol hexadecyl ether(BRIJ 58P, x=20), polyethylene glycol octadecyl ether (BRIJ 72, x=2),polyethylene glycol octadecyl ether (BRIJ 76, x=10), polyethylene glycoloctadecyl ether (BRIJ® 78P, x=20), polyethylene glycol oleyl ether (BRIJ92V, x=2), and polyoxyl 10 oleyl ether (BRIJ 97, x=10);poly-D-glucosamine (chitosan); chlorobutanol; cholesterol;diethanolamine; digitonin; dimethylsulfoxide (DMSO), ethylenediaminetetraacetic acid (EDTA); glyceryl monosterate; lanolin alcohols; mono-and di-glycerides; monoethanolamine; nonylphenol polyoxyethylene ether(NP-40); octylphenoxypolyethoxyethanol (NONIDET NP-40 from Amresco);ethyl phenol poly (ethylene glycol ether)^(n), n=1 l (NONIDET P40 fromRoche); octyl phenol ethylene oxide condensate with about 9 ethyleneoxide units (NONIDET P40); IGEPAL CA 630 ((octyl phenoxy)polyethoxyethanol; structurally same as NONIDET NP-40); oleic acid;oleyl alcohol; polyethylene glycol 8000; polyoxyl 20 cetostearyl ether;polyoxyl 35 castor oil; polyoxyl 40 hydrogenated castor oil; polyoxyl 40stearate; polyoxyethylene sorbitan monolaurate (polysorbate 20, orTWEEN-20; polyoxyethylene sorbitan monooleate (polysorbate 80, orTWEEN-80); propylene glycol diacetate; propylene glycol monostearate;protamine sulfate; proteolytic enzymes; sodium dodecyl sulfate (SDS);sodium monolaurate; sodium stearate; sorbitan derivatives (SPAN), e.g.,sorbitan monopalmitate (SPAN 40), sorbitan monostearate (SPAN 60),sorbitan tristearate (SPAN 65), sorbitan monooleate (SPAN 80), andsorbitan trioleate (SPAN 85);2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosa-hexaene (squalene);stachyose; stearic acid; sucrose; surfactin (lipopeptide antibiotic fromBacillus subtilis); dodecylpoly(ethyleneglycolether)9 (THESIT) MW 582.9;octyl phenol ethylene oxide condensate with about 9-10 ethylene oxideunits (TRITON X-100); octyl phenol ethylene oxide condensate with about7-8 ethylene oxide units (TRITON X-114); tris(2-hydroxyethyl)amine(trolamine); and emulsifying wax.

In certain adjuvant compositions, the adjuvant is a cytokine Acomposition of the present invention can comprise one or more cytokines,chemokines, or compounds that induce the production of cytokines andchemokines, or a polynucleotide encoding one or more cytokines,chemokines, or compounds that induce the production of cytokines andchemokines Examples include, but are not limited to, granulocytemacrophage colony stimulating factor (GM-CSF), granulocyte colonystimulating factor (G-CSF), macrophage colony stimulating factor(M-CSF), colony stimulating factor (CSF), erythropoietin (EPO),interleukin 2 (IL-2), interleukin-3 (IL-3), interleukin 4 (IL-4),interleukin 5 (IL-5), interleukin 6 (IL-6), interleukin 7 (IL-7),interleukin 8 (IL-8), interleukin 10 (IL-10), interleukin 12 (IL-12),interleukin 15 (IL-15), interleukin 18 (IL-18), interferon alpha (IFNα),interferon beta (IFNβ), interferon gamma (IFNγ), interferon omega(IFNΩ), interferon tau (IFNτ), interferon gamma inducing factor I(IGIF), transforming growth factor beta (TGF-β), RANTES (regulated uponactivation, normal T-cell expressed and presumably secreted), macrophageinflammatory proteins (e.g., MIP-1 alpha and M3P-1 beta), Leishmaniaelongation initiating factor (LEIF), and Flt-3 ligand.

In certain compositions of the present invention, the polynucleotideconstruct may be complexed with an adjuvant composition comprising(±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(syn-9-tetradecenyloxy)-1-propanaminiumbromide (GAP-DMORIE). The composition may also comprise one or moreco-lipids, e.g., 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE),1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPyPE), and/or1,2-dimyristoyl-glycer-3-phosphoethanolamine (DMPE). An adjuvantcomposition comprising GAP-DMORIE and DPyPE at a 1:1 molar ratio isreferred to herein as VAXFECTIN adjuvant. See, e.g., PCT Publication No.WO 00/57917.

In other embodiments, the polynucleotide itself may function as anadjuvant as is the case when the polynucleotides of the invention arederived, in whole or in part, from bacterial DNA. Bacterial DNAcontaining motifs of unmethylated CpG-dinucleotides (CpG-DNA) triggersinnate immune cells in vertebrates through a pattern recognitionreceptor (including toll receptors such as TLR 9) and thus possessespotent immunostimulatory effects on macrophages, dendritic cells andB-lymphocytes. See, e.g., Wagner, H., Curr. Opin. Microbiol. 5:62-69(2002); Jung, J. et al., J. Immunol. 169: 2368-73 (2002); see alsoKlinman, D. M. et al., Proc. Natl. Acad. Sci. U.S.A. 93:2879-83 (1996).Methods of using unmethylated CpG-dinucleotides as adjuvants aredescribed in, for example, U.S. Pat. Nos. 6,207,646, 6,406,705 and6,429, 199.

The ability of an adjuvant to increase the immune response to an antigenis typically manifested by a significant increase in immune-mediatedprotection. For example, an increase in humoral immunity is typicallymanifested by a significant increase in the titer of antibodies raisedto the antigen, and an increase in T-cell activity is typicallymanifested in increased cell proliferation, or cellular cytotoxicity, orcytokine secretion. An adjuvant may also alter an immune response, forexample, by changing a primarily humoral or Th₂ response into aprimarily cellular, or Th_(I) response.

Nucleic acid molecules and/or polynucleotides of the present invention,e.g., plasmid DNA, mRNA, linear DNA or oligonucleotides, may besolubilized in any of various buffers. Suitable buffers include, forexample, phosphate buffered saline (PBS), normal saline, Tris buffer,and sodium phosphate (e.g., 150 mM sodium phosphate). Insolublepolynucleotides may be solubilized in a weak acid or weak base, and thendiluted to the desired volume with a buffer. The pH of the buffer may beadjusted as appropriate. In addition, a pharmaceutically acceptableadditive can be used to provide an appropriate osmolarity. Suchadditives are within the purview of one skilled in the art. For aqueouscompositions used in vivo, sterile pyrogen-free water can be used. Suchformulations will contain an effective amount of a polynucleotidetogether with a suitable amount of an aqueous solution in order toprepare pharmaceutically acceptable compositions suitable foradministration to a human.

Compositions of the present invention can be formulated according toknown methods. Suitable preparation methods are described, for example,in Remington's Pharmaceutical Sciences, 16th Edition, A. Osol, ed., MackPublishing Co., Easton, Pa. (1980), and Remington's PharmaceuticalSciences, 19th Edition, A. R. Gennaro, ed., Mack Publishing Co., Easton,Pa. (1995). Although the composition may be administered as an aqueoussolution, it can also be formulated as an emulsion, gel, solution,suspension, lyophilized form, or any other form known in the art. Inaddition, the composition may contain pharmaceutically acceptableadditives including, for example, diluents, binders, stabilizers, andpreservatives.

The following examples are included for purposes of illustration onlyand are not intended to limit the scope of the present invention, whichis defined by the appended claims.

EXAMPLES Materials and Methods

The following materials and methods apply generally to all the examplesdisclosed herein. Specific materials and methods are disclosed in eachexample, as necessary.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology (including PCR), vaccinology, microbiology,recombinant DNA, and immunology, which are within the skill of the art.Such techniques are explained fully in the literature. See, for example,Molecular Cloning A Laboratory Manual, 2nd Ed., Sambrook et al., ed.,Cold Spring Harbor Laboratory Press: (1989); DNA Cloning, Volumes I andII (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed.,1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization(B. D. Hames & S. J. Higgins eds. 1984); Transcription And Translation(B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I.Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRLPress, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984);the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); GeneTransfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds.,1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154and 155 (Wu et al. eds.), Immunochemical Methods In Cell And MolecularBiology (Mayer and Walker, eds., Academic Press, London, 1987); and inAusubel et al., Current Protocols in Molecular Biology, John Wiley andSons, Baltimore, Md. (1989).

Gene Construction

Constructs of the present invention are constructed based on thesequence information provided herein or in the art utilizing standardmolecular biology techniques, including, but not limited to, thefollowing. First, a series complementary oligonucleotide pairs of 80-90nucleotides each in length and spanning the length of the construct aresynthesized by standard methods. These oligonucleotide pairs aresynthesized such that upon annealing, they form double strandedfragments of 80-90 base pairs, containing cohesive ends. Thesingle-stranded ends of each pair of oligonucleotides are designed toanneal with a single-stranded end of an adjacent oligonucleotide duplex.Several adjacent oligonucleotide pairs prepared in this manner areallowed to anneal, and approximately five to six adjacentoligonucleotide duplex fragments are then allowed to anneal together viathe cohesive single stranded ends. This series of annealedoligonucleotide duplex fragments is then ligated together and clonedinto a suitable plasmid, such as the TOPO® vector available fromInvitrogen Corporation, Carlsbad, Calif. The construct is then sequencedby standard methods. Constructs prepared in this manner, comprising 5 to6 adjacent 80 to 90 base pair fragments ligated together, i.e.,fragments of about 500 base pairs, are prepared, such that the entiredesired sequence of the construct is represented in a series of plasmidconstructs. The inserts of these plasmids are then cut with appropriaterestriction enzymes and ligated together to form the final construct.The final construct is then cloned into a standard bacterial cloningvector, and sequenced. The oligonucleotides and primers referred toherein can easily be designed by a person of skill in the art based onthe sequence information provided herein and in the art, and such can besynthesized by any of a number of commercial nucleotide providers, forexample Retrogen, San Diego, Calif., and GENEART, Regensburg, Germany.

Plasmid Vectors

Constructs of the present invention can be inserted, for example, intoeukaryotic expression vectors VR1012 or VR10551. These vectors are builton a modified pUC18 background (see Yanisch-Perron, C., et al. Gene33:103-119 (1985)), and contain a kanamycin resistance gene, the humancytomegalovirus immediate early promoter/enhancer and intron A, and thebovine growth hormone transcription termination signal, and a polylinkerfor inserting foreign genes. See Hartikka, J., et al., Hum. Gene Ther.7:1205-1217 (1996). However, other standard commercially availableeukaryotic expression vectors may be used in the present invention,including, but not limited to: plasmids pcDNA3, pHCMV/Zeo, pCR3.1,pEF1/H is, pIND/GS, pRc/HCMV2, pSV40/Zeo2, pTRACER-HCMV, pUB6/V5-His,pVAX1, and pZeoSV2 (available from Invitrogen, San Diego, Calif.), andplasmid pCI (available from Promega, Madison, Wis.).

An optimized backbone plasmid, termed VR10551, has minor changes fromthe VR1012 backbone described above. The VR10551 vector is derived fromand similar to VR1012 in that it uses the human cytomegalovirusimmediate early (hCMV-IE) gene enhancer/promoter and 5′ untranslatedregion (UTR), including the hCMV-IE Intron A. The changes from theVR1012 to the VR10551 include some modifications to the multiple cloningsite, and a modified rabbit β globin 3′ untranslatedregion/polyadenylation signal sequence/transcriptional terminator hasbeen substituted for the same functional domain derived from the bovinegrowth hormone gene.

Plasmid DNA Purification

Plasmid DNA may be transformed into competent cells of an appropriateEscherichia coli strain (including but not limited to the DH5α strain)and highly purified covalently closed circular plasmid DNA was isolatedby a modified lysis procedure (Horn, N. A., et al., Hum. Gene Ther.6:565-573 (1995)) followed by standard double CsCl-ethidium bromidegradient ultracentrifugation (Sambrook, J., et al., Molecular Cloning: ALaboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press,Plainview, N.Y. (1989)). Alternatively, plasmid DNAs are purified usingGiga columns from Qiagen (Valencia, Calif.) according to the kitinstructions. All plasmid preparations were free of detectablechromosomal DNA, RNA and protein impurities based on gel analysis andthe bicinchoninic protein assay (Pierce Chem. Co., Rockford Ill.).Endotoxin levels were measured using Limulus Amebocyte Lysate assay(LAL, Associates of Cape Cod, Falmouth, Mass.) and were less than 0.6Endotoxin Units/mg of plasmid DNA. The spectrophotometric A₂₆₀/A₂₈₀ratios of the DNA solutions were typically above 1.8. Plasmids wereethanol precipitated and resuspended in an appropriate solution, e.g.,150 mM sodium phosphate (for other appropriate excipients and auxiliaryagents, see U.S. patent application Publication 2002/0019358, publishedFeb. 14, 2002). DNA was stored at −20° C. until use. DNA was diluted bymixing it with 300 mM salt solutions and by adding appropriate amount ofUSP water to obtain 1 mg/ml plasmid DNA in the desired salt at thedesired molar concentration.

Plasmid Expression in Mammalian Cell Lines

The expression plasmids are analyzed in vitro by transfecting theplasmids into a well characterized mouse melanoma cell line (VM-92, alsoknown as UM-449). See, e.g., Wheeler, C. J., Sukhu, L., Yang, G., Tsai,Y., Bustamente, C. Felgner, P. Norman, J & Manthorpe, M. “Converting anAlcohol to an Amine in a Cationic Lipid Dramatically Alters the Co-lipidRequirement, Cellular Transfection Activity and the Ultrastructure ofDNA-Cytofectin Complexes,” Biochim. Biophys. Acta. 1280:1-11 (1996).Other well-characterized human cell lines can also be used, e.g. MRC-5cells, American Type Culture Collection (ATCC) Accession No. CCL-171 orhuman rhabdomyosarcoma cell line RD (ATCC CCL-136). The transfection isperformed using cationic lipid-based transfection procedures well knownto those of skill in the art. Other transfection procedures are wellknown in the art and may be used, for example electroporation andcalcium chloride-mediated transfection (Graham F. L. and A. J. van derEb Virology 52:456-67 (1973)). Following transfection, cell lysates andculture supernatants of transfected cells are evaluated to comparerelative levels of expression of herpes simplex virus antigen proteins.The samples are assayed by western blots and ELISAs, using commerciallyavailable polyclonal and/or monoclonal antibodies (available, e.g., fromResearch Diagnostics Inc., Flanders N.J.), so as to compare both thequality and the quantity of expressed antigen.

In addition to plasmids encoding single herpes simplex virus proteins,single plasmids which contain two or more herpes simplex virus codingregions are constructed according to standard methods. For example, apolycistronic construct, where two or more herpes simplex virus codingregions are transcribed as a single transcript in eukaryotic cells maybe constructed by separating the various coding regions with IRESsequences. Alternatively, two or more coding regions may be insertedinto a single plasmid, each with their own promoter sequence.

Codon Optimization Algorithm

The following is an outline of the algorithm used to derive humancodon-optimized sequences of herpes simplex antigens.

Back Translation

Starting with the amino acid sequence, one can either (a) manuallybacktranslate using the human codon usage table on the world wide web atkazusa.or.ip/codon/

Homo sapiens [gbpri]: 55194 CDS's (24298072 codons)

Fields: [triplet] [frequency: per thousand] ([number])

TABLE 6 UUU 17.1(415589) UCU 14.7(357770) UAU 12.1(294182) UGU10.0(243198) UUC 20.6(500964) UCC 17.6(427664) UAC 15.5(377811) UGC12.2(297010) UUA 7.5(182466) UCA 12.0(291788) UAA 0.7(17545) UGA1.5(36163) UUG 12.6(306793) UCG 4.4(107809) UAG 0.6(13416) UGG12.7(309683) CUU 13.0(315804) CCU 17.3(419521) CAU 10.5(255135) CGU4.6(112673) CUC 19.8(480790) CCC 20.1(489224) CAC 15.0(364828) CGC10.7(259950) CUA 7.8(189383) CCA 16.7(405320) CAA 12.0(292745) CGA6.3(152905) CUG 39.8(967277) CCG 6.9(168542) CAG 34.1(827754) CGG11.6(281493) AUU 16.1(390571) ACU 13.0(315736) AAU 16.7(404867) AGU11.9(289294) AUC 21.6(525478) ACC 19.4(471273) AAC 19.5(473208) AGC19.3(467869) AUA 7.7(186138) ACA 15.1(366753) AAA 24.1(585243) AGA11.5(278843) AUG 22.2(538917) ACG 6.1(148277) AAG 32.2(781752) AGG11.4(277693) GUU 11.0(266493) GCU 18.6(451517) GAU 21.9(533009) GGU10.8(261467) GUC 14.6(354537) GCC 28.4(690382) GAC 25.6(621290) GGC22.5(547729) GUA 7.2(174572) GCA 16.1(390964) GAA 29.0(703852) GGA16.4(397574) GUG 28.4(690428) GCG 7.5(181803) GAG 39.9(970417) GGG16.3(396931) * Coding GC 52.45% 1st letter GC 56.04% 2nd letter GC42.37% 3rd letter GC 58.93% (Table as of Nov. 6, 2003)Coding GC 52.45% 1st letter GC 56.04% 2nd letter GC 42.37% 3rd letter GC58.93% (Table as of Nov. 6, 2003)

-   -   Or (b) log on to the world wide web at syntheticgenes.com and        use the backtranslation tool, as follows:

(1) Under Protein tab, paste amino acid sequence;

(2) Under download codon usage tab, highlight homo sapiens and thendownload CUT.

TABLE 7 UUU 17.1(415589) UCU 14.7(357770) UAU 12.1(294182) UGU10.0(243198) UUC 20.6(500964) UCC 17.6(427664) UAC 15.5(377811) UGC12.2(297010) UUA 7.5(182466) UCA 12.0(291788) UAA 0.7(17545) UGA1.5(36163) UUG 12.6(306793) UCG 4.4(107809) UAG 0.6(13416) UGG12.7(309683) CUU 13.0(315804) CCU 17.3(419521) CAU 10.5(255135) CGU4.6(112673) CUC 19.8(480790) CCC 20.1(489224) CAC 15.0(364828) CGC10.7(259950) CUA 7.8(189383) CCA 16.7(405320) CAA 12.0(292745) CGA6.3(152905) CUG 39.8(967277) CCG 6.9(168542) CAG 34.1(827754) CGG11.6(281493) AUU 16.1(390571) ACU 13.0(315736) AAU 16.7(404867) AGU11.9(289294) AUC 21.6(525478) ACC 19.4(471273) AAC 19.5(473208) AGC19.3(467869) AUA 7.7(186138) ACA 15.1(366753) AAA 24.1(585243) AGA11.5(278843) AUG 22.2(538917) ACG 6.1(148277) AAG 32.2(781752) AGG11.4(277693) GUU 11.0(266493) GCU 18.6(451517) GAU 21.9(533009) GGU10.8(261467) GUC 14.6(354537) GCC 28.4(690382) GAC 25.6(621290) GGC22.5(547729) GUA 7.2(174572) GCA 16.1(390964) GAA 29.0(703852) GGA16.4(397574) GUG 28.4(690428) GCG 7.5(181803) GAG 39.9(970417) GGG16.3(396931) (Table as of Nov. 6, 2003)

(3) Hit Apply button.

(4) Under Optimize TAB, open General TAB.

(5) Check use only most frequent codon box.

(6) Hit Apply button.

(7) Under Optimize TAB, open Motif TAB.

(8) Load desired cloning restriction sites into bad motifs; load anyundesirable sequences, such as Pribnow Box sequences (TATAA), Chisequences (GCTGGCGG), and restriction sites into bad motifs.

(9) Under Output TAB, click on Start box. Output will include sequence,motif search results (under Report TAB), and codon usage report.

The program did not always use the most frequent codon for amino acidssuch as cysteine proline, and arginine. To change this, go back to theEdit CUT TAB and manually drag the rainbow colored bar to 100% for thedesired codon. Then re-do start under the Output TAB.

The use of CGG for arginine can lead to very high GC content, so AGA canbe used for arginine as an alternative. The difference in codon usage is11.6 per thousand for CGG vs. 11.5 per thousand for AGA.

Splice Donor and Acceptor Site Search

(1) Log on to Berkeley Drosophila Genome Project Website at on the worldwide web at fruitfly.org/seg tools/spice.html\.

(2) Check boxes for Human or other and both splice sites.

(3) Select minimum scores for 5′ and 3′ splice sites between 0 and 1.

-   -   Used the default setting at 0.4 where:    -   Default minimum score is 0.4, where:

% splice % false sites recognized positives Human 5′ Splice sites 93.2%5.2% Human 3′ Splice sites 83.8% 3.1%

(4) Paste in sequence.

(5) Submit.

(6) Based on predicted donors or acceptors, change the individual codonsuntil the sites are no longer predicted.

Add in 5′ and 3′ Sequences.

On the 5′ end of the gene sequence, the restriction enzyme site andKozak sequence (gccacc) was added before ATG. On 3′ end of the sequence,tca was added following the stop codon (tga on opposite strand) and thena restriction enzyme site. The GC content and Open Reading Frames werethen checked in SEC Central.

Preparation of Vaccine Formulations

Plasmid constructs comprising codon-optimized and non-codon-optimizedcoding regions encoding gD, VP 11/12, VP13/14 and/or VP22; oralternatively coding regions (either codon-optimized or non-codonoptimized) encoding various herpes simplex virus proteins or fragments,variants or derivatives either alone or as fusions with a carrierprotein, e.g., HBcAg, as well as various controls, e.g., empty vector,are formulated with the poloxamer CRL 1005 and BAK (Benzalkoniumchloride 50% solution, available from Ruger Chemical Co. Inc.) by thefollowing methods. Specific final concentrations of each component ofthe formulae are described in the following methods, but for any ofthese methods, the concentrations of each component maybe varied bybasic stoichiometric calculations known by those of ordinary skill inthe art to make a final solution having the desired concentrations.

For example, the concentration of CRL 1005 is adjusted depending on, forexample, transfection efficiency, expression efficiency, orimmunogenicity, to achieve a final concentration of between about 1mg/ml to about 75 mg/ml, for example, about 1 mg/ml, about 2 mg/ml,about 3 mg/ml, about 4 mg/ml, about 5 mg/ml, about 6.5 mg/ml, about 7mg/ml, about 7.5 mg/ml, about 8 mg/ml, about 9 mg/ml, about 10 mg/ml,about 15 mg/ml, about 20 mg/ml, about 25 mg/ml, about 30 mg/ml, about 35mg/ml, about 40 mg/ml, about 45 mg/ml, about 50 mg/ml, about 55 mg/ml,about 60 mg/ml, about 65 mg/ml, about 70 mg/ml, or about 75 mg/ml of CRL1005.

Similarly the concentration of DNA is adjusted depending on manyfactors, including the amount of a formulation to be delivered, the ageand weight of the subject, the delivery method and route and theimmunogenicity of the antigen being delivered. In general, formulationsof the present invention are adjusted to have a final concentration fromabout 1 ng/ml to about 30 mg/ml of plasmid (or other polynucleotide).For example, a formulation of the present invention may have a finalconcentration of about 1 ng/ml, about 5 ng/ml, about 10 ng/ml, about 50ng/ml, about 100 ng/ml, about 500 ng/ml, about 1 μg/ml, about 5 μg/ml,about 10 μg/ml, about 50 μg/ml, about 200 μg/ml, about 400 μg/ml, about600 μg/ml, about 800 μg/ml, about 1 mg/ml, about 2 mg/ml, about 2.5,about 3 mg/ml, about 3.5, about 4 mg/ml, about 4.5, about 5 mg/ml, about5.5 mg/ml, about 6 mg/ml, about 7 mg/ml, about 8 mg/ml, about 9 mg/ml,about 10 mg/ml, about 20 mg/ml, or about 30 mg mg/ml of a plasmid.

Certain formulations of the present invention include a cocktail ofplasmids of the present invention, e.g., comprising coding regionsencoding herpes simplex virus proteins gD, VP 11/12, VP13/14 and/or VP22and optionally, plasmids encoding immunity enhancing proteins, e.g.,cytokines Various plasmids desired in a cocktail are combined togetherin PBS or other diluent prior to the addition to the other ingredients.Furthermore, plasmids may be present in a cocktail at equal proportions,or the ratios may be adjusted based on, for example, relative expressionlevels of the antigens or the relative immunogenicity of the encodedantigens. Thus, various plasmids in the cocktail may be present in equalproportion, or up to twice or three times as much of one plasmid may beincluded relative to other plasmids in the cocktail.

Additionally, the concentration of BAK may be adjusted depending on, forexample, a desired particle size and improved stability. Indeed, incertain embodiments, formulations of the present invention include CRL1005 and DNA, but are free of BAK. In general BAK-containingformulations of the present invention are adjusted to have a finalconcentration of BAK from about 0.05 mM to about 0.5 mM. For example, aformulation of the present invention may have a final BAK concentrationof about 0.05 mM, 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM or 0.5 mM.

The total volume of the formulations produced by the methods below maybe scaled up or down, by choosing apparatus of proportional size.Finally, in carrying out any of the methods described below, the threecomponents of the formulation, BAK, CRL 1005, and plasmid DNA, may beadded in any order. In each of these methods described below the term“cloud point” refers to the point in a temperature shift, or othertitration, at which a clear solution becomes cloudy, i.e., when acomponent dissolved in a solution begins to precipitate out of solution.

Thermal Cycling of a Pre-Mixed Formulation

This example describes the preparation of a formulation comprising 0.3mM BAK, 7.5 mg/ml CRL 1005, and 5 mg/ml of DNA in a total volume of 3.6ml. The ingredients are combined together at a temperature below thecloud point and then the formulation is thermally cycled to roomtemperature (above the cloud point) several times.

A 1.28 mM solution of BAK is prepared in PBS, 846 μl of the solution isplaced into a 15 ml round bottom flask fitted with a magnetic stirringbar, and the solution is stirred with moderate speed, in an ice bath ontop of a stirrer/hotplate (hotplate off) for 10 minutes. CRL 1005 (27μl) is then added using a 100 μl positive displacement pipette and thesolution is stirred for a further 60 minutes on ice. Plasmids comprisingcodon-optimized coding regions, and optionally, additional plasmidscomprising codon-optimized or non-codin-optimized coding regionsencoding, e.g., additional herpes simplex virus proteins, and or otherproteins, e.g., cytokines, are mixed together at desired proportions inPBS to achieve 6.4 mg/ml total DNA. This plasmid cocktail is added dropwise, slowly, to the stirring solution over 1 min using a 5 ml pipette.The solution at this point (on ice) is clear since it is below the cloudpoint of the poloxamer and is further stirred on ice for 15 min. The icebath is then removed, and the solution is stirred at ambient temperaturefor 15 minutes to produce a cloudy solution as the poloxamer passesthrough the cloud point.

The flask is then placed back into the ice bath and stirred for afurther 15 minutes to produce a clear solution as the mixture is cooledbelow the poloxamer cloud point. The ice bath is again removed and thesolution stirred at ambient temperature for a further 15 minutes.Stirring for 15 minutes above and below the cloud point (total of 30minutes), is defined as one thermal cycle. The mixture is cycled sixmore times. The resulting formulation may be used immediately, or may beplaced in a glass vial, cooled below the cloud point, and frozen at −80°C. for use at a later time.

Animal Immunizations

The immunogenicity of the various herpes simplex virus expressionproducts encoded by the codon-optimized polynucleotides described hereinare initially evaluated based on each plasmid's ability to mount animmune response in vivo. Plasmids are tested individually and incombinations by injecting single constructs as well as multipleconstructs. Immunizations are initially carried out in animals, such asmice, rabbits, goats, sheep, non-human primates, or other suitableanimal, by intramuscular (IM) or intradermal (ID) injections. Serum iscollected from immunized animals, and the antigen specific antibodyresponse is quantified by ELISA assay using purified immobilized antigenproteins in a protein—immunized subject antibody—anti-species antibodytype assay, according to standard protocols. The tests of immunogenicityfurther include measuring antibody titer, neutralizing antibody titer,T-cell proliferation, T-cell secretion of cytokines, cytolytic T cellresponses, and by direct enumeration of antigen specific CD4+ and CD8+T-cells. Correlation to protective levels of the immune responses inhumans are made according to methods well known by those of ordinaryskill in the art.

DNA Formulations

Plasmid DNA is formulated with a poloxamer. Alternatively, plasmid DNAis prepared and dissolved at a concentration of about 0.1 mg/ml to about10 mg/ml, preferably about 1 mg/ml, in PBS with or withouttransfection-facilitating cationic lipids, e.g., DMRIE/DOPE at a 4:1DNA:lipid mass ratio. Alternative DNA formulations include 150 mM sodiumphosphate instead of PBS, adjuvants, e.g., VAXFECTIN adjuvant at a 4:1DNA: VAXFECTIN adjuvant mass ratio, mono-phosphoryl lipid A (detoxifiedendotoxin) from S. Minnesota (MPL) and trehalosedicorynomycolateAF(TDM), in 2% oil (squalene)-TWEEN-80 polysorbate 80-water (MPL+TDM,available from Sigma/Aldrich, St. Louis, Mo., (catalog #M6536)), asolubilized mono-phosphoryl lipid A formulation (AF, available fromCorixa), or(±)-N-(3-Acetoxypropyl)-N,N-dimethyl-2,3-bis(octyloxy)-1-propanaminiumchloride (compound #VC1240) (see Shriver, J. W. et al., Nature415:331-335 (2002), and P.C.T. Publication No. WO 02/00844 A2.

Animal Immunizations

Plasmid constructs comprising codon-optimized and non-codon-optimizedcoding regions encoding gD, VP 11/12, VP13/14 and/or VP22; oralternatively coding regions (either codon-optimized or non-codonoptimized) encoding various herpes simplex virus proteins or fragments,variants or derivatives either alone or as fusions with a carrierprotein, e.g., HBcAg, as well as various controls, e.g., empty vector,are injected into BALB/c mice as single plasmids or as cocktails of twoor more plasmids, as either DNA in PBS or formulated with thepoloxamer-based delivery system: 2 mg/ml DNA, 3 mg/ml CRL 1005, and 0.1mM BAK. Groups of 10 mice are immunized three times, at biweeklyintervals, and serum is obtained to determine antibody titers to each ofthe antigens. Groups are also included in which mice are immunized witha trivalent preparation, containing each of the three plasmid constructsin equal mass.

The immunization schedule is as follows:

Day 3 Pre-bleed

Day 0 Plasmid injections, intramuscular, bilateral in rectus femoris,5-50 μg/leg

Day 21 Plasmid injections, intramuscular, bilateral in rectus femoris,5-50 μg/leg

Day 49 Plasmid injections, intramuscular, bilateral in rectus femoris,5-50 μg/leg

Day 59 Serum collection

Serum antibody titers are determined by ELIS A with recombinantproteins, peptides or transfection supernatants and lysates fromtransfected VM-92 cells live, inactivated, or lysed virus.

Immunization of Mice with Vaccine Formulations Using a VAXFECTINAdjuvant

VAXFECTIN adjuvant (a 1:1 molar ratio of the cationic lipid VC1052 andthe neutral co-lipid DPyPE) is a synthetic cationic lipid formulationwhich enhances antibody titers against when administered with DNAintramuscularly to mice.

VAXFECTIN adjuvant mixtures are prepared by mixing chloroform solutionsof VC1052 cationic lipid with chloroform solutions of DpyPE neutralco-lipid. Dried films are prepared in 2 ml sterile glass vials byevaporating the chloroform under a stream of nitrogen, and placing thevials under vacuum overnight to remove solvent traces. Each vialcontains 1.5 μmole each of VC1052 and DPyPE. Liposomes are prepared byadding sterile water followed by vortexing. The resulting liposomesolution is mixed with DNA at a phosphate mole:cationic lipid mole ratioof 4:1.

Plasmid constructs comprising codon-optimized and non-codon-optimizedcoding regions encoding gD, VP 11/12, VP13/14 and/or VP22; oralternatively coding regions (either codon-optimized or non-codonoptimized) encoding various herpes simplex virus proteins or fragments,variants or derivatives either alone or as fusions with a carrierprotein, e.g., HBcAg, as well as various controls, e.g., empty vector,are mixed together at desired proportions in PBS to achieve a finalconcentration of 1.0 mg/ml. The plasmid cocktail, as well as thecontrols, are formulated with VAXFECTIN adjuvant. Groups of 5 BALB/cfemale mice are injected bilaterally in the rectus femoris muscle with50 μl of DNA solution (100 μl total/mouse), on days 1 and 21 and 49 witheach formulation. Mice are bled for serum on days 0 (prebleed), 20(bleed 1), and 41 (bleed 2), and 62 (bleed 3), and up to 40 weekspost-injection. Antibody titers to the various herpes simplex virusproteins encoded by the plasmid DNAs are measured by ELISA.

Cytolytic T-cell responses are measured as described in Hartikka et al.“Vaxfectin Enhances the Humoral Response to Plasmid DNA-encodedAntigens,” Vaccine 19:1911-1923 (2001). Standard ELISPOT technology isused for the CD4+ and CD8+ T-cell assays.

Production of Antisera in Animals

Plasmid constructs comprising codon-optimized and non-codon-optimizedcoding regions encoding gD, VP 11/12, VP13/14 and/or VP22; oralternatively coding regions (either codon-optimized or non-codonoptimized) encoding various herpes simplex virus proteins or fragments,variants or derivatives either alone or as fusions with a carrierprotein, e.g., HBcAg, as well as various controls, e.g., empty vector,are prepared according to the immunization scheme described above andinjected into a suitable animal for generating polyclonal antibodies.Serum is collected and the antibody titered as above.

Monoclonal antibodies are also produced using hybridoma technology(Kohler, et al., Nature 256:495 (1975); Kohler, et al., Eur. J. Immunol.6:511 (1976); Kohler, et al., Eur. J. Immunol. 6:292 (1976); Hammerling,et al., in Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y.,(1981), pp. 563-681. In general, such procedures involve immunizing ananimal (preferably a mouse) as described above. The splenocytes of suchmice are extracted and fused with a suitable myeloma cell line. Anysuitable myeloma cell line may be employed in accordance with thepresent invention; however, it is preferable to employ the parentmyeloma cell line (SP2O), available from the American Type CultureCollection, Rockville, Md. After fusion, the resulting hybridoma cellsare selectively maintained in HAT medium, and then cloned by limitingdilution as described by Wands et al., Gastroenterology 80:225-232(1981), incorporated herein by reference in its entirety. The hybridomacells obtained through such a selection are then assayed to identifyclones which secrete antibodies capable of binding the various herpessimplex virus proteins.

Alternatively, additional antibodies capable of binding to herpessimplex virus proteins described herein may be produced in a two-stepprocedure through the use of anti-idiotypic antibodies. Such a methodmakes use of the fact that antibodies are themselves antigens, and that,therefore, it is possible to obtain an antibody which binds to a secondantibody. In accordance with this method, various herpes simplexvirus-specific antibodies are used to immunize an animal, preferably amouse. The splenocytes of such an animal are then used to producehybridoma cells, and the hybridoma cells are screened to identify cloneswhich produce an antibody whose ability to bind to the herpes simplexvirus protein-specific antibody can be blocked by the cognate herpessimplex virus protein. Such antibodies comprise anti-idiotypicantibodies to the herpes simplex virus protein-specific antibody and canbe used to immunize an animal to induce formation of further herpessimplex virus-specific antibodies.

It will be appreciated that Fab and F(ab′)2 and other fragments of theantibodies of the present invention maybe used. Such fragments aretypically produced by proteolytic cleavage, using enzymes such as papain(to produce Fab fragments) or pepsin (to produce F(ab′)2 fragments).Alternatively, gD, VP 11/12, VP13/14 and/or VP22 binding fragments canbe produced through the application of recombinant DNA technology orthrough synthetic chemistry.

It may be preferable to use “humanized” chimeric monoclonal antibodies.Such antibodies can be produced using genetic constructs derived fromhybridoma cells producing the monoclonal antibodies described above.Methods for producing chimeric antibodies are known in the art. See, forreview, Morrison, Science 229:1202 (1985); 01, et al., BioTechniques4:214 (1986); Cabilly, et al., U.S. Pat. No. 4,816,567; Taniguchi, etal., EP 171496; Morrison, et al., EP 173494; Neuberger, et al., WO8601533; Robinson, et al, WO 8702671; Boulianne, et al., Nature 312:643(1984); Neuberger, et al., Nature 314:268 (1985).

These antibodies are used, for example, in diagnostic assays, as aresearch reagent, to screen animals for identification of the vaccine'seffectiveness, or to further immunize animals to generate herpes simplexvirus-specific anti-idiotypic antibodies. Non-limiting examples of usesfor anti-herpes simplex virus antibodies include use in Western blots,ELISA (competitive, sandwich, and direct), immunofluorescence,immunoelectron microscopy, radioimmunoassay, immunoprecipitation,agglutination assays, immunodiffusion, immunoelectrophoresis, andepitope mapping (Weir, D. Ed. Handbook of Experimental Immunology, 4thed. Vols. I and II, Blackwell Scientific Publications (1986)).

Mucosal Vaccination and Electrically Assisted Plasmid Delivery MucosalDNA Vaccination

Plasmid constructs comprising codon-optimized and non-codon-optimizedcoding regions encoding gD, VP 11/12, VP13/14 and/or VP22; oralternatively coding regions (either codon-optimized or non-codonoptimized) encoding various herpes simplex virus proteins or fragments,variants or derivatives either alone or as fusions with a carrierprotein, e.g., HBcAg, as well as various controls, e.g., empty vector,(100 μg/50 μl total DNA) are delivered to BALB/c mice at 0, 2 and 4weeks via i.m., intranasal (i.n.), intravenous (i.v.), intravaginal(i.vag.), intrarectal (i.r.) or oral routes. The DNA is deliveredunformulated or formulated with the cationic lipids DMRIE/DOPE (DD) orGAP-DLRIE/DOPE (GD). As endpoints, serum IgG titers against the variousherpes simplex virus antigens are measured by ELISA and splenic T-cellresponses are measured by antigen-specific production of IFN-gamma andIL-4 in ELISPOT assays. Standard chromium release assays are used tomeasure specific cytotoxic T lymphocyte (CTL) activity against thevarious herpes simplex virus antigens. Tetramer assays are used todetect and quantify antigen specific T-cells, with quantification beingconfirmed and phenotypic characterization accomplished by intracellularcytokine staining. In addition, IgG and IgA responses against thevarious herpes simplex virus antigens are analyzed by ELISA of vaginalwashes.

Electrically-Assisted Plasmid Delivery

In vivo gene delivery may be enhanced through the application of briefelectrical pulses to injected tissues, a procedure referred to herein aselectrically-assisted plasmid delivery (EAPD). See, e.g., Aihara, H. &Miyazaki, J. Nat. Biotechnol. 16:867-70 (1998); Mir, L. M. et al., Proc.Natl. Acad. Sci. USA 96:4262-67 (1999); Hartikka, J. et al., Mol. Ther.4:407-15 (2001); and Mir, L. M. et al.; Rizzuto, G. et al., Hum GeneTher 11:1891-900 (2000); Widera, G. et al, J. of Immuno. 164:4635-4640(2000). The use of electrical pulses for cell electropermeabilizationhas been used to introduce foreign DNA into prokaryotic and eukaryoticcells in vitro. Cell permeabilization can also be achieved locally, invivo, using electrodes and optimal electrical parameters that arecompatible with cell survival.

The electroporation procedure can be performed with variouselectroporation devices. These devices include external plate typeelectrodes or invasive needle/rod electrodes and can possess twoelectrodes or multiple electrodes placed in an array. Distances betweenthe plate or needle electrodes can vary depending upon the number ofelectrodes, size of target area and treatment subject.

The TriGrid needle array is a three electrode array comprising threeelongate electrodes in the approximate shape of a geometric triangle.Needle arrays may include single, double, three, four, five, six or moreneedles arranged in various array formations. The electrodes areconnected through conductive cables to a high voltage switching devicethat is connected to a power supply.

The electrode array is placed into the muscle tissue, around the site ofnucleic acid injection, to a depth of approximately 3 mm to 3 cm. Thedepth of insertion varies depending upon the target tissue and size ofpatient receiving electroporation. After injection of foreign nucleicacid, such as plasmid DNA, and a period of time sufficient fordistribution of the nucleic acid, square wave electrical pulses areapplied to the tissue. The amplitude of each pulse ranges from about 100volts to about 1500 volts, e.g., about 100 volts, about 200 volts, about300 volts, about 400 volts, about 500 volts, about 600 volts, about 700volts, about 800 volts, about 900 volts, about 1000 volts, about 1100volts, about 1200 volts, about 1300 volts, about 1400 volts, or about1500 volts or about 1-1.5 kV/cm, based on the spacing betweenelectrodes. Each pulse has a duration of about 1 μs to about 1000 μs,e.g., about 1 μs, about 10 μs, about 50 μs, about 100 μs, about 200 μs,about 300 μs, about 400 μs, about 500 μs, about 600 μs, about 700 μs,about 800 μs, about 900 μs, or about 1000 μs, and a pulse frequency onthe order of about 1-10 Hz. The polarity of the pulses may be reversedduring the electroporation procedure by switching the connectors to thepulse generator. Pulses are repeated multiple times. The electroporationparameters (e.g. voltage amplitude, duration of pulse, number of pulses,depth of electrode insertion and frequency) will vary based on targettissue type, number of electrodes used and distance of electrodespacing, as would be understood by one of ordinary skill in the art.

Immediately after completion of the pulse regimen, subjects receivingelectroporation can be optionally treated with membrane stabilizingagents to prolong cell membrane permeability as a result of theelectroporation. Examples of membrane stabilizing agents include, butare not limited to, steroids (e.g. dexamethasone, methylprednisone andprogesterone), angiotensin II and vitamin E. A single dose ofdexamethasone, approximately 0.1 mg per kilogram of body weight, shouldbe sufficient to achieve a beneficial affect.

EAPD techniques such as electroporation can also be used for plasmidscontained in liposome formulations. The liposome-plasmid suspension isadministered to the animal or patient and the site of injection istreated with a safe but effective electrical field generated, forexample, by a TriGrid needle array. The electroporation may aid inplasmid delivery to the cell by destabilizing the liposome bilayer sothat membrane fusion between the liposome and the target cellularstructure occurs. Electroporation may also aid in plasmid delivery tothe cell by triggering the release of the plasmid, in highconcentrations, from the liposome at the surface of the target cell sothat the plasmid is driven across the cell membrane by a concentrationgradient via the pores created in the cell membrane as a result of theelectroporation.

To test the effect of electroporation on therapeutic protein expressionin non-human primates, male or female rhesus monkeys are given either 2or 6 i.m. injections of plasmid constructs comprising codon-optimizedand non-codon-optimized coding regions encoding gD, VP 11/12, VP13/14and/or VP22; or alternatively coding regions (either codon-optimized ornon-codon optimized) encoding various herpes simplex virus proteins orfragments, variants or derivatives either alone or as fusions with acarrier protein, e.g., HBcAg, as well as various controls, e.g., emptyvector, (0.1 to 10 mg DNA total per animal). Target muscle groupsinclude, but are not limited to, bilateral rectus fermoris, cranialtibialis, biceps, gastrocenemius or deltoid muscles. The target area isshaved and a needle array, comprising between 4 and 10 electrodes,spaced between 0.5-1.5 cm apart, is implanted into the target muscle.Once injections are complete, a sequence of brief electrical pulses areapplied to the electrodes implanted in the target muscle using an IchorTGP-2 pulse generator. The pulses have an amplitude of approximately120-200V. The pulse sequence is completed within one second. During thistime, the target muscle may make brief contractions or twitches. Theinjection and electroporation may be repeated.

Sera are collected from vaccinated monkeys at various time points. Asendpoints, serum IgG titers against the various herpes simplex virusantigens are measured by ELISA and PBMC T-cell proliferative responsesare measured by antigen-specific production of IFN-gamma and IL-4 inELISPOT assays or by tetramer assays to detect and quantify antigenspecific T-cells, with quantification being confirmed and phenotypiccharacterization accomplished by intracellular cytokine stainingStandard chromium release assays are used to measure specific cytotoxicT lymphocyte (CTL) activity against the various TV antigens.

Vaccine Construction and Evaluation

UL46, 47, and 49 open reading frames (ORFs) from seven to eight HSV-2strains isolated from primary genital infections were PCR-amplified andsequenced. The consensus was used for vaccine design.

Plasmids were constructed by cloning synthetic, full-length,codon-optimized UL46, UL47, or UL49 DNA into backbone plasmid VR1012.

Codon-optimized UL46, 47, and 49 constructs were compared with fulllength wild-type UL46, 47 and 49 by co-transfecting Cos-7 cells with thedifferent individual constructs plus full-length cDNA encoding HLAA*0201, A*0101, or B*0702. CD8⁺ T-cell clones known to react to UL46,UL47, or UL49 epitopes were incubated with the transfected cells, withan ELISA for IFN-γ secretion employed as readout.

DNA vaccines were formulated with PBS, with VAXFECTIN adjuvant at 1 μgDNA/1.09 μg VAXFECTIN adjuvant, or with poloxamer, such as, but notlimited to, CRL1005, at 1 μg DNA/1.5 μg poloxamer in PBS.

Animals and Immunization Regimen

Female, 4-8 week old BALB/c mice (10 per group) were immunized with 50μg DNA in 50 μL by IM injection in bilateral quadriceps (100μg/immunization) on Days 0, 14, and 28.

Sera were collected from each animal Days −1 (pre-bleed), 13, 27, and42. Splenocytes were isolated at terminal sacrifice Day 42.

EUSA Evaluation of Antibody Response

Antigens for ELISA were derived from recombinant full-length tegumentproteins made by transiently transfecting VM92 cells with the vaccineconstructs and collecting supernatants.

Sera were serially diluted in TBS with 0.1% BSA and 0.05% TWEEN-20polysorbate 20. Standard reagents measured antigen-specific IgG byabsorbance at OD₄₅₀ nm. A positive (Day 42) serum pool for each antigenwas run on every ELISA plate. An exponential curve was generated fromvalues in the mid portion of dilution vs. OD₄₅₀ (corrected forbackground absorbance). Antibody titers for each animal at a time pointwere calculated as the mean titer determined from the individualdilutions (calculated from the standard curve). If the OD₄₅₀ for alldilutions of experimental sera were very low, the titer was assigned as1:1.

Evaluation of Cellular Response by ELISPOT Assay

IFN-γ secretion by T-cells was assessed by ELISPOT assay with standardreagents. Plates were read by computer.

Splenocytes (0.5−1×10⁶ cells/well in duplicate wells) from individualanimals were stimulated using peptide pools (18-24 13-mer peptidesoverlapping by 9 amino acids, at 0.42-0.56 μg/ml each), or positive (ConA) or negative (media) controls. Pooled responder splenocytes were alsotested with single 13-mer peptides at 10 μg/ml.

Peptides found to produce responses were further evaluated using shorter(9-11 aa) peptides, dose-response curves, and/or CD4⁺ or CD8⁺ responders(negative selection, Miltenyi, >80% pure) back-mixed with naivesplenocyte APC. Plates with too-numerous-to-count spots were arbitrarilyassigned 103 spots.

Discussion

HSV-2 UL46, UL47, and UL49 DNA vaccines are immunogenic in BALB/c (H-2d)mice.

H-2d CD8+ epitopes were found with all vaccines, and CD4⁺ epitopes werefound for UL46 and UL49.

CD8⁺ splenocytes respond to peptides at concentrations from 10⁻³ to 10⁻⁶μM; CD4⁺ splenocytes require peptide concentrations of 10⁻¹ μM orhigher.

Cellular responses in BALB/c mice are greatest for UL47, followed byUL49 and UL46, when expressed as SFU/106 splenocytes.

Relative antibody titers are UL49>UL47>UL46.

The poloxamer-based formulation boosted humoral immunity on Day 42 forall three tegument DNA vaccines by 3-5-fold compared to no adjuvant.Formulations based on Vaxfectin™ boosted antibody responses to UL46 andUL49 DNA about 2-fold by Day 42 relative to no adjuvant.

Poloxamer significantly boosted cellular responses to UL47 DNA vaccine(p=0.03), but not to UL46 or UL49 DNA vaccines. VAXFECTIN adjuvant hadno statistically significant adjuvant effect on any tested vaccine(p>0.05).

Cellular immunity to three HSV-2 tegument proteins was detectable afterDNA vaccination. Responses to both UL47 and UL49 were particularlystrong. Due to difficulties with peptide synthesis, 19% of the UL46peptides were missing in the assay, as compared to 1 or 2 peptidesmissing for assays involving UL47 and UL49. Nonetheless, multiple CD4⁺and CD8⁺ epitopes have been identified that should assist animalpathogenesis studies in BALB/c mice.

The adjuvant effects of poloxamer and VAXFECTIN adjuvant basedformulations were moderate and inconsistent between antigens. Adjuvanteffect may be more apparent with a lower dose of antigen, or when usedin higher species.

Identify the HSV-2 Genes of Interest.

HSV-2 encodes ˜85 proteins (Roizman, B., Knipe, D. M., Whitley, R. J.,Herpes simplex viruses, in Fields Virology, D. M. Knipe, Howley, P. M.,Editor. 2007, Lippincott, Williams, and Wilkins: Philadelphia, p.2501-2602). The pDNA approach is limited to one or a few ORFs. Criteriafor choosing ORFs include quick recognition of infected cells,immunodominance (within-person and within-population), antiviraleffector functions, and localization to lesions.

Tegument proteins UL46, UL47, UL49, and UL7 are CD8+ antigens (Koelle,D. M., et al., CD8 CTL from genital herpes simplex lesions: recognitionof viral tegument and immediate early proteins and lysis of infectedcutaneous cells. Journal of Immunology, 2001. 166: p. 4049-4058). CD8⁺T-cells specific for UL47 and UL49 are abundant enough to be detected by“direct” PBMC staining with HLA-peptide tetramers (Koelle, D. M., etal., Expression of cutaneous lymphocyte-associated antigen byCD8+T-cells specific for a skin-tropic virus. Journal of ClinicalInvestigation, 2002. 110: p. 537-548). CD8⁺ cells specific forglycoprotein, capsid, or immediate early proteins are less abundant, andhave never been detected in “direct” PBMC staining (Koelle,unpublished). Studies of up to 95 independently derived HSV-2-specificCD8+ clones per subject showed that responses to tegument proteins wereimmunodominant (Koelle, D. M., Liu Z., McClurkan C. L., Cevallos R. C.,Vieira J., Hosken N. A., Meseda C. A., Snow D. C., Wald A., Corey L.,Immunodominance among herpes simplex virus-specific CD8 T-cellsexpressing a tissue-specific homing receptor. Proc Natl Acad Sci USA,2003. 100: p. 12899-12904).

We then measured human CD8⁺ responses to 48 HSV-2 ORFs (57% of thetotal) by making 5,230 synthetic peptides covering these ORFs. CD8⁺T-cells from peripheral blood were probed by IFN-γ ELISPOT as thereadout (Hosken, N., McGowan P., Meier A., Koelle D. M., Sleath P.,Wegener F., Elliott M., Grabstein L., Posavad C., Corey L., Diversity ofthe CD8+T cell response to herpes simpolex virus type 2 proteins amongpersons with genital herpes. Journal of Virology, 2006. 80: p.5509-5515). Among 24 HSV-2-infected subjects, 50% recognized thetegument proteins UL46 and UL47. Responses to UL49 were slightly lower(˜40%).

A trial of a HSV-2 therapeutic vaccine comprised of an HLAA*0201-restricted epitope in envelope glycoprotein B and an adjuvant isbeing conducted. At baseline (n=42 persons), the population prevalenceof responses to an HLA A*0201 epitope in gB is 45% compared to 68% foran A*0201 epitope in the tegument protein UL47 (p=0.012, Fisher's exacttest).

One advantage of tegument-specific CD8⁺ T-cells, over cells specific forimmediate early, capsid, or envelope proteins, is that tegument-specificCD8⁺ T-cells can kill target cells immediately after they are infected.This is due to recognition of virion input protein, as proven in CTLassays with the transcriptional blocker actinomycin D, or target cellssensitized with UV-treated virus (Koelle, D. M., et al., CD8 CTL fromgenital herpes simplex lesions: recognition of viral tegument andimmediate early proteins and lysis of infected cutaneous cells. Journalof Immunology, 2001. 166: p. 4049-4058).

Recognition of Processed Tegument Input Protein Bypasses the ImmuneEvasion by HSV TAP Inhibitor Protein ICP47.

In addition, tetramer in situ stain of human skin biopsies inhealing/post-healing phases of genital HSV-2 lesions, show thattetramer-specific CD8+ T-cells localize adjacent to HSV-2-infected cellsduring recurrences, and monitor the region of peripheral nerve endingsat the dermal-epidermal junction after healing (Zhu, J., Koelle, D. M.,Cao, J., Vezquez, J., Huang, M X., Hladik, F., Wald, A., Corey, L.,Peripheral virus-specific CD8+T cells contiguous to sensory nerveendings limit HSV-2 reactivation in human genital skin. Journal ofExperimental Medicine, 2007. epub Feb. 26, 2007: p. epub Feb. 26, 2007).

Taken together, within-population and within-subject dominance, highabsolute numerical levels, localization to lesional and post-healingskin, antiviral effector functions, and immediate recognition ofinfected cells all argue that HSV-2 tegument proteins are rationaltargets for CD8+-directed therapeutic approach to decreasing HSV-2lesions, symptoms, and shedding.

Determine the Amino Acid Sequence for Candidate Vaccin Tegument HSV-2Genes.

We sequenced tegument proteins UL46, UL47, and UL49 in circulating NorthAmerican HSV-2 strains. We isolated HSV-2 from persons with documentedprimary genital herpes (Ashley, R. A., et al., Comparison of Westernblot (immunoblot) and glycoprotein G-specific immunoblot for detectingantibodies to herpes simplex types 1 and 2 in human sera. Journal ofClinical Microbiology, 1988. 26: p. 662-667), passaged them minimally invitro, PCR-amplified tegument genes with an accurate polymerase, andsequenced them bi-directionally (Martin, E., Koelle D M, Byrd B, Huang ML, Vieira J, Corey L, Wald A, Sequence-based methods for identifyingepidemiologically linked herpes simplex virus type 2 strains. J ClinMicrobiol, 2006. 44(7): p. 2541-6). We identified many loci at which allthe wild-type strains shared the same coding difference from HG52(Dolan, A., et al., The genome sequence of herpes simplex virus type 2.Journal of Virology, 1998. 72: p. 2010-2021). At other loci, there werepolymorphic amino acids that differed between wild-type strains. Forthese, we picked the prevalent alleles for our vaccine composition.(Table 8)

TABLE 8 Selected coding polymorphisms in eight wild-type HSV-2 isolates.Amino acid (AA) numbers per HG52 nomenclature (Dolan, A., et al., Thegenome sequence of herpes simplex virus type 2. Journal of Virology,1998. 72: p. 2010-2021). Table entries list HG52 AA followed bywild-type. Vaccine sequences VR2145, VR2144, and VR2143 are also shown.UL46 amino acid strain 78 110 364 425 436 471 474 587 594 613 634 637638 644 672 673 346 TR CF delA EK PL RQ LD LP RP RG 2589 AS TR CF HP PLRQ LD LP RP RG 2899 KN TR CF delA EK GS PL RQ LD LP RP RG 7124 TR CFdelA GS PL RQ LD LP RP RG 7566 KN TR CF delA EK GS PL RQ LD LP RP RG10875 KN TR CF delA EK GS PL RQ LD LP RP RG 11449 TR AV VG CF delA EK GSPL RQ LD LP RP RG 16293 KN TR CF delA EK GS PL RQ LD LP RP RG VR2145 A KR A V F H delA K S L Q D P P G UL47 amino acid UL49 amino acid strain 338 69 82 156 172 177 529 strain 73-74 76 87 94 134 346 VA PS PA SP 346SE insert SA SP 2589 NS PA SP RH 2589 SE insert DN RH SP 2899 VG NS PASP 2899 7566 VG NS PA SP 7566 10875 VG RQ NS PA SP 10875 11449 VG NS PASP 11449 16293 VG NS PA SP 16293 VR2144 V G P R S A P R VR2143 no insertD R S S

We were concerned that CD8+ T-cell epitopes might be under selectivepressure to mutate and “escape” CD8⁺ CTL, as proven for HIV (Nolan, D.,I. James, and S. Mallal, HIV/AIDS. HIV: experiencing the pressures ofmodern life. Science, 2005. 307(5714): p. 1422-4). HSV-2 has an accurateDNA polymerase, but mutations do occur during acyclovir therapy(Czartoski, T., et al., Fulminant, acyclovir-resistant, herpes simplexvirus type 2 hepatitis in an immunocompetent woman. J Clin Microbiol,2006. 44(4): p. 1584-6). We sequenced more than 100 additional wild-typeHSV-2 strains, focusing on known CD8⁺ epitopes regions in UL46, UL47,and UL49. We found no coding polymorphisms in or near CD8+ epitopes(Koelle, D. M., et al., CD8 CTL from genital herpes simplex lesions:recognition of viral tegument and immediate early proteins and lysis ofinfected cutaneous cells. Journal of Immunology, 2001. 166: p.4049-4058; Koelle, D. M., Liu Z., McClurkan C. L., Cevallos R. C.,Vieira J., Hosken N. A., Meseda C. A., Snow D. C., Wald A., Corey L.,Immunodominance among herpes simplex virus-specific CD8 T-cellsexpressing a tissue-specific homing receptor. Proc Natl Acad Sci USA,2003. 100: p. 12899-12904) in UL46 or UL47, or in or near CD4⁺ epitopes(Koelle, D. M., et al., Recognition of herpes simplex virus type 2tegument proteins by CD4 T cells infiltrating human genital herpeslesions. Journal of Virology, 1998. 72: p. 7476-7483; Posavad, C M., etal., T cell immunity to herpes simplex virus in seronegative persons:silent infection or acquired immunity. Journal of Immunology, 2003. 170:p. 4380-4388; Koelle, D. M., et al., Tegument-specific, virus-reactiveCD4 T-cells localize to the cornea in herpes simplex virus interstitialkeratitis in humans. Journal of Virology, 2000. 74: p. 10930-10938) inany protein.

In contrast, there is marked heterogeneity in the HLA B*0702-restrictedCD8⁺ epitope in UL49 (AA 49-57). While 70% of isolates have the majoritysequence RPRGEVREFL, 29% have the minority RPMREVRFL, and 1% have therare RPRGKVRFL. Our immune studies (Koelle, D. M., Liu, C., Byrd, B.,Sette, A., Sidney, J., Wald, A. HSV-2 VP22 sequences from wild-typeisolates that escape a dominant CD8 CTL response in linkage with apolymorphism at an adjacent casein kinase II substrate domain, in 30thInternational Herpesvirus Workshop, 2005. Turku, Finland), in brief,show that while all 3 variants bind well to recombinant HLA B*0702[IC₅₀<2 nM, assays as per (Southwood, S., et al., Several common HLA-DRtypes share largely overlapping peptide binding repertoires. J Immunol,1998. 160(7): p. 3363-73)], CD8⁺ T-cells specific for the “majority” arenot cross-reactive with “minority” or “rare” variants. Vaccination withthe 70% consensus “majority” would miss 30% of circulating strains.

Optimize the Genes for Protein Expression and Stability, Synthesizethem, and Clone them into a pDNA Backbone.

After establishing the AA sequences for the pDNA vaccines, we usedproprietary codon optimization algorithms with the goals of higheukaryotic expression. The genes were synthesized by GeneArt. Versionsof UL46, UL47, and UL49 were made with or without the gD₁ epitope tagQPELAPEDPED. Each was cloned into plasmid VR1012 (Hartikka, J., et al.,An improved plasmid DNA expression vector for direct injection intoskeletal muscle. Hum Gene Ther, 1996. 7(10): p. 1205-17). VR1012 encodeskanamycin resistance, and contains a promoter/enhancer and intron A ofCMV immediate early gene 1, and a bovine growth hormone-basedterminator. VR1012 was chosen because 1) it achieves high levelexpression in many cells, animal species, and tissues; 2) there has beenno stability problems in in-house studies; 3) high plasmid yields areobtained in E. coli; and most importantly 4) VR1012-based products arein clinical trials. The inserts were sequence verified to an averageredundancy of 4-fold.

VAXFECTIN adjuvant was used for intramuscular injection studies.VAXFECTIN adjuvant is an equimolar mixture of VC1052((±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(myristoleyloxy)-1-propanaminium bromide) and DPyPE(diphytanoylphosphatidyl-ethanolamine) (Hartikka, J., et al., Animproved plasmid DNA expression vector for direct injection intoskeletal muscle. Hum Gene Ther, 1996. 7(10): p. 1205-17). A lipid filmwas prepared by mixing chloroform solutions of VC1052 and DPyPE in glassvials, evaporating the chloroform, and vacuum packing. At the time ofvaccination, the lipid film was reconstituted to 2.18 mg/mL with 1 mL of0.9% saline. Vaccines were prepared at a final pDNA (phosphate):cationic lipid molar ratio of 4:1 by adding an equal volume of lipid topDNA (2 mg/mL in 0.9% saline, 20 mM sodium phosphate). Reconstitutedvaccine was held at room temperature and used within 60 minutes.

Initial experiments expressed the gD-tagged versions of UL46, UL47, andUL49 by transient transfection of VM92 cells (Kumar, S., et al., A DNAvaccine encoding the 42 kDa C-terminus of merozoite surface protein 1 ofPlasmodium falciparum induces antibody, interferon-gamma and cytotoxic Tcell responses in rhesus monkeys: immuno-stimulatory effects ofgranulocyte macrophage-colony stimulating factor. Immunol Lett, 2002.81(1): p. 13-24). Immunoblots showed bands at the predicted MW (notshown). All subsequent experiments used the full-length tegumentconstructs without the epitope tag. We used human CD8+ CTL clonesspecific for UL46, UL47, and UL49 (Koelle, D. M., et al., CD8 CTL fromgenital herpes simplex lesions: recognition of viral tegument andimmediate early proteins and lysis of infected cutaneous cells. Journalof Immunology, 2001. 166: p. 4049-4058; Koelle, D. M., Liu Z., McClurkanC. L., Cevallos R. C., Vieira J., Hosken N. A, Meseda C. A., Snow D. C.,Wald A., Corey L., Immunodominance among herpes simplex virus-specificCD8 T-cells expressing a tissue-specific homing receptor. Proc Natl AcadSci USA, 2003. 100: p. 12899-12904) to establish that the pDNA vaccinesencoded proteins that are processed and presented to CD8+ T-cells. COS-7cells were co-transfected with (1) candidate vaccine plasmid and (2)cDNA encoding a specific human HLA class I α-chain. The human HLA classI heavy chains form a functional heterodimer with non-human primate(COS-7 cell) β₂ microglobulin (β₂M). If the vaccine construct encodes aprotein that can be processed to antigenic peptides, some HLA classI-β₂M heterodimers will translocate to the cell surface loaded withHSV-2 peptides. After two days, human CD8⁺ T cell clones specific forrelevant tegument proteins were added. T-cell activation was detected byIFN-γ ELISA of the supernatant (Koelle, D. M., et al., CD8 CTL fromgenital herpes simplex lesions: recognition of viral tegument andimmediate early proteins and lysis of infected cutaneous cells. Journalof Immunology, 2001. 166: p. 4049-4058; Koelle, D. M., et al.,Expression of cutaneous lymphocyte-associated antigen by CD8+T-cellsspecific for a skin-tropic virus. Journal of Clinical Investigation,2002. 110: p. 537-548). Specific responses were detected (FIG. 3). Tcell clones did not recognize COS-7 cells transfected with HLA class IcDNA alone, HSV-2 plasmid DNA alone, or HSV-2 DNA plus the “wrong” HLA(not shown).

The proteins encoded by the candidate vaccines were also recognized byhuman anti-HSV antibodies. To make vaccine-encoded protein, VM92 cells(Kumar, S., et al., A DNA vaccine encoding the 42 kDa C-terminus ofmerozoite surf ace protein 1 of Plasmodium falciparum induces antibody,interferon-gamma and cytotoxic T cell responses in rhesus monkeys:immuno-stimulatory effects of granulocyte macrophage-colony stimulatingfactor. Immunol Lett, 2002. 81(1): p. 13-24) were transfected with thevaccine plasmids, and supernatants collected. These were used as antigen(1:5 dilution) to coat ELISA plates. Pooled sera from HSV-2 seropositiveindividuals bound recombinant tegument proteins (FIG. 4), while pooledsera from HSV-2 seronegative individuals did not. These tests show thatbona fide HSV-2-specific T-cells and antibodies recognizevaccine-encoded HSV-2 tegument proteins.

Measure Immune Responses to HSV-2 Tegument Plasmids Alone or inCombination in Mice.

We chose the female BALB/c mouse (H-2^(d)) so we could combineimmunogenicity and protective efficacy tests. The only previously knownHSV-2 CD8+ epitope in BALB/c mice is in protein ICP27 (Haynes, J.,Arrington J, Dong L, Braun R P, Payne L G, Potent protective cellularimmune responses generated by a DNA vaccine encoding HSV-2 ICP27 and theE. coli heat labile enterotoxin. Vaccine, 2006. 24(23): p. 5016-26).Several type-common regions of gD are CD4+ epitopes in these animals(BenMohamed, L., et al., Identification of novel immunodominantCD4+Th1-type T-cell peptide epitopes from herpes simplex virusglycoprotein D that confer protective immunity. J Virol, 2003. 77(17):p. 9463-73). BALB/c mice are very susceptible to intravaginal infectionwith HSV-2 (Lopez, C., Genetics of natural resistance to herpes virusinfections in mice. Nature, 1975. 258: p. 1352-1353).

Humoral response. We immunized mice with 100 μg pDNA on days 0, 14, and28 as 50 μg IM per quadriceps. We compared VAXFECTIN adjuvant and a CRL1005 poloxamer as shown in 6,844,001 as Example 1 (Selinsky, C, et al.,A DNA-based vaccine for the prevention of humancytomegahvirus-associated diseases. Hum Vaccin, 2005.1(1): p. 16-23) asadjuvants versus PBS. As we have focused on VAXFECTIN adjuvant, andresults (titers, speed to antibody, titers at each time point forantibody, and IFN-γ sfu/10⁶ splenocytes for T-cells) were generallysimilar for the adjuvants and PBS at this high pDNA dose, only VAXFECTINadjuvant data are shown.

Every mouse produced antibodies against the relevant protein by thesecond immunization (FIGS. 20A through 20I). Antibody titers weresignificantly higher from one measurement to the next (p<0.03, pairedtwo-tailed t-test) for each vaccine and every time-point comparison. Wealso verified that vaccine-elicited antibodies bound to a whole HSV-2lysate (FIG. 4). These data again show the plasmids encode bona fideHSV-2 proteins.

CD8⁺ and CD4⁺ Responses to Tegument DNA Vaccines in BALB/c Mice.

Overlapping peptides 13 AA long, offset by four AA, were synthesized tomatch the predicted vaccine sequences (Table 8). Initial assays usedpeptide pools (18-24 peptides/pool, concentration for each peptide 0.5μg/mL). Splenocytes from individual mice (FIG. 6A through 6C), harvestedtwo weeks after the third immunization, were assayed. The readout wasIFN-γ ELISPOT (Haynes, J., Arrington J, Dong L, Braun R P, Payne L G,Potent protective cellular immune responses generated by a DNA vaccineencoding HSV-2 ICP27 and the E. coli heat labile enterotoxin. Vaccine,2006. 24(23): p. 5016-26). Responses to pools were summed for eachanimal to give cumulative responses, expressed as spot forming units(sfu)/10⁶ splenocytes. For UL47 and UL49, responses were higher thanfrom naive mice (p<0.01, two-tailed t-test). For UL46, responses werenot statistically different from naive (p=0.37), due to high responsesin two naive mice. However, testing of single peptides from UL46 stilldisclosed antigenic peptides.

The individual peptides in positive pools were tested in follow-upELISPOT, and in each case, single or neighboring overlapping peptideswere positive. We used overlap regions (when present) and MHC-peptideepitope prediction algorithms (Bui, H. H., et al., Automated generationand evaluation of specific MHC binding predictive tools: ARB matrixapplications. Immunogenetics, 2005; Parker, K. C., M. A. Bednarek, andJ. E. Coligan, Scheme for ranking potential HLA-A2 binding peptidesbased on independent binding of individual peptide side-chains. Journalof Immunology, 1994. 152: p. 163-168; Rammensee, H., et al., SYFPEITHI:database for MHC ligands and peptide motifs. Immunogenetics, 1999. 50:p. 213-319) to pick shorter peptides for further tests. We used negativeselection with magnetic bead-conjugated antibodies to enrich CD4⁺ orCD8⁺ splenocytes, and back-mixed these with naive congenic splenocytesas APC. In IFN-γ ELISPOT, potent CD8⁺ epitopes were found for eachvaccine protein. CD4⁺ responses were detected in UL46 and UL49. CD4⁺responses were generally weaker than CD8⁺ responses when quantified assfu/10⁶ splenocytes or EC₅₀ (the concentration giving 50% of the maximumresponse) (FIG. 21). Some CD8⁺ epitopes were active at 10⁻¹² M (FIG.21). Such potent CD8+ epitopes typically bind tightly to relevant MCHclass I molecules. The IC₅₀ for binding UL46 183-191 (KYAAAVAGL) toH-2K^(d) was 9.91 nM (very tight binding) (Sette, A., et al., A roadmapfor the immunomics of category A-C pathogens. Immunity, 2005. 22(2): p.155-61). Overall, the tegument protein vaccines elicited high avidity,and often multi-epitope and combined (CD4⁺ and CD8⁺) T cell responses.

HSV-2-Infected Mice Generate T Cells Against Tegument Protein Epitopes.

T cells primed in vivo by HSV-2 infection would be boosted byvaccination. In this context, it was important to test iftegument-specific T cells were primed in vivo by HSV-2 infection, aswell as by vaccine (above). We infected BALB/c mice with an attenuatedHSV-2 strain 333 variant deficient in thymidine kinase (333tk-)(Milligan, G. N. and D. I. Bernstein, Generation of humoral responsesagainst herpes simplex virus type 2 in the murine female genital tract.Virology, 1995. 206: p. 234-241). Mice were made susceptible tointravaginal infection by subcutaneous Depo-provera (progestin) 6 daysbefore infection. Splenocytes from day 14 (FIGS. 22 and 23) mice showedT cell responses to CD8+ tegument epitopes previously discovered usingpDNA vaccines (above). In both humans and mice, tegument proteins UL46,UL47, and UL49 are processed and presented via the MHC class I pathwayduring viral infection.

Tegument Vaccines Provide Partial Protection Against Lethal IntravaginalChallenge with HSV-2.

“CD8⁺-only” vaccines can protect mice from lethal intracerebral orfootpad HSV-1 challenge (Blaney, J. E., et al., Immunization with asingle major histocompatibility class I-restricted cytotoxicT-lymphocyte recognition epitope of herpes simplex virus type 2 confersprotective immunity. Journal of Virology, 1998. 72: p. 9567-9574; On, M.T., Orgun, N. N., Wilson, C. B., Way, S. S., Cutting edge: recombinantlisteria monocytogenes expressing a single immune-dominant peptideconfers immunity to herpes simplex virus-1 infection. Journal ofImmunology, 2007. 178: p. In Press Apr. 15, 2007 edition), but havenever been studied in the intravaginal HSV-2 model. We found thatunivalent tegument vaccines provided partial protection in theintravaginal model. We used the virulent HSV-2 strain 186 (Nishiyama, Y.and F. Rapp, Latency in vitro using irradiated herpes simplex virus. JGen Virol, 1981. 52(Pt 1): p. 113-9) for lethal challenge. 3×10³ pfu.The endpoints were measured twice a day, day 14 survival, and vaginalHSV-2 titers on day 1-5. All-dacron swabs were placed into 1 mL PCRbuffer, extracted, and analyzed for HSV-2 DNA copy number byhigh-throughput real-time PCR as described (Ryncarz, A. J., et al.,Development of a high throughput quantitative assay for detecting HSVDNA in clinical samples. Journal of Clinical Microbiology, 1999. 37: p.1941-1947). Positive vaccine controls were intravaginal infection with10⁶ pfu of attenuated HSV-2 333tk-(after Depo-provera), or threeinjections of a truncated gD₂ pDNA vaccine (please see below). Negativecontrol was empty plasmid. pDNA vaccines were given as 3 doses on days0, 14, and 28 at 100 μg/dose IM in PBS. Mice were challenged 14 daysafter vaccination with 50 times LD₅₀ (50×(3×10³)=1.5×10⁵ pfu) afterDepo-provera.

HSV-2 333tk- and gD₂ protected all animals (FIG. 24). UL47 pDNA provided44% protection (4 of 9 animals), with possible slight protection forUL49. The UL47 and UL49 survivors were confirmed to have been infectedby ELISA using whole HSV-2 lysate; they had much higher OD₄₅₀ values(data not shown) than could be explained by immunity to the immunizingconstruct alone (FIGS. 20A through 2I). The tegument vaccines werenon-sterilizing: HSV-2 replication occurred in the vagina afterchallenge (FIG. 24). The gD₂ vaccine (below) did lead to a measurablereduction in HSV-2 replication (FIG. 24). The clinical severity scorewas reduced after UL47, UL49, and gD₂ vaccination.

gD₂ Shows Minimal Sequence Variation and is an Effective PreventativeVaccine that Lowers HSV-2 Replication.

We sequenced six wild-type gD₂ genes. We found few changes from HG52:one had V169A and a second had V353A and L375P changes. No changes weredetected in known gD₂ CD8+ or neutralizing epitopes (Koelle, D. M., LiuZ., McClurkan C. L., Cevallos R. C., Vieira J., Hosken N. A, Meseda C.A., Snow D. C., Wald A, Corey L., Immunodominance among herpes simplexvirus-specific CD8 T-cells expressing a tissue-specific homing receptor.Proc Natl Acad Sci USA, 2003. 100: p. 12899-12904; Tigges, M. A., etal., Human CD8+ herpes simplex virus-specific cytotoxic T lymphocyteclones recognize diverse virion protein antigens. Journal of Virology,1992. 66: p. 1622-1634; Spear, P. G., R. J. Eisenberg, and G. H. Cohen.Three classes of surface receptors for alphaherpesvirus entry. Virology,2000. 275: p. 1-8.). Our candidate pDNA gD₂ vaccine, VR2139, encodes AA1-340 of gD₂ using the HG52 sequence. AA 341-393 were omitted becausethey contain a leader and transmembrane domain. Humoral responses weredetected by ELISA using commercially available gD₁ as coating antigenand a commercially available mAb against a type-common gD epitope as acalibrator (FIG. 25). Cellular responses were detected with overlapping13-mer peptides exactly as described above for tegument proteins. Afterthree vaccinations of 100 μg gD₂ pDNA vaccine on days 0, 14, and 28 withVAXFECTIN adjuvant, brisk humoral and total splenocyte IFN-γ ELISPOTresponses were noted in most animals (FIG. 25). Survival, clinicalseverity and intravaginal HSV-2 DNA viral load benefits were describedabove.

SUMMARY

CD8⁺ T cell responses control HSV infection in mice and humans in theskin and ganglia. Tegument proteins are important targets of the CD8+human immune response to HSV-2 (Koelle, D. M., et al., CD8 CTL fromgenital herpes simplex lesions: recognition of viral tegument andimmediate early proteins and lysis of infected cutaneous cells. Journalof Immunology, 2001. 166: p. 4049-4058; Koelle, D. M., et al.,Recognition of herpes simplex virus type 2 tegument proteins by CD4 Tcells infiltrating human genital herpes lesions. Journal of Virology,1998. 72: p. 7476-7483; Posavad, C. M., et al., T cell immunity toherpes simplex virus in seronegative persons: silent infection oracquired immunity. Journal of Immunology, 2003. 170: p. 4380-4388;Koelle, D. M., Liu Z., McClurkan C. L., Cevallos R. C., Vieira J.,Hosken N. A, Meseda C. A., Snow D. C., Wald A., Corey L.,Immunodominance among herpes simplex virus-specific CD8 T-cellsexpressing a tissue-specific homing receptor. Proc Natl Acad Sci USA,2003. 100: p. 12899-12904; Koelle, D. M., et al., Tegument-specific,virus-reactive CD4 T-cells localize to the cornea in herpes simplexvirus interstitial keratitis in humans. Journal of Virology, 2000. 74:p. 10930-10938; Verjans, G. M., et al., Intraocular T cells of patientswith herpes simplex (HSV)-induced acute retinal necrosis recognize HSVtegument proteins VP11/12 and VP13/14. Journal of Infectious Diseases,2000. 182: p. 923-927). DNA vaccines encoding HSV-2 tegument proteinswere found to stimulate CD8⁺, CD4⁺, and antibody responses, and selectedunivalent vaccines were partially protective in an intravaginalchallenge model.

Other Embodiments

The foregoing description is considered as illustrative only of theprinciples of the invention. Further, since numerous modifications andchanges will readily occur to those skilled in the art, it is notdesired to limit the invention to the exact construction and process asdescribed above. Accordingly, all suitable modifications and equivalentsmay be resorted to falling within the scope of the invention as definedby the claims that follow. The words “comprise,” “comprising,”“include,” “including,” and “includes” when used in this specificationand in the following claims are intended to specify the presence ofstated features, integers, components, or steps, but they do notpreclude the presence or addition of one or more other features,integers, components, steps, or groups thereof.

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

All patent documents and references cited herein are incorporated byreference as if fully set forth.

What is claimed is:
 1. A DNA composition comprising an isolatedpolynucleotide comprising SEQ ID NOs: 9, 11 and 12; and an adjuvant. 2.The DNA composition of claim 1, wherein the adjuvant is a cationic lipidwith one or more co-lipids.
 3. The DNA composition of claim 2, whereinthe cationic lipid is(±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(syn-9-tetradeceneyloxy)-1-propanaminiumbromide) (GAP-DMORIE).
 4. The DNA composition of claim 2, wherein thecationic lipid is GAP-DMORIE and the co-lipid is1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPyPE).